key: cord-010046-7hlgjiqp
authors: Harvey, David J.
title: Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2003–2004
date: 2008-09-29
journal: Mass Spectrom Rev
DOI: 10.1002/mas.20192
sha: 
doc_id: 10046
cord_uid: 7hlgjiqp

This review is the third update of the original review, published in 1999, on the application of matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings the topic to the end of 2004. Both fundamental studies and applications are covered. The main topics include methodological developments, matrices, fragmentation of carbohydrates and applications to large polymeric carbohydrates from plants, glycans from glycoproteins and those from various glycolipids. Other topics include the use of MALDI MS to study enzymes related to carbohydrate biosynthesis and degradation, its use in industrial processes, particularly biopharmaceuticals and its use to monitor products of chemical synthesis where glycodendrimers and carbohydrate–protein complexes are highlighted. © 2009 Wiley Periodicals, Inc., Mass Spec Rev 28:273–361, 2009

This review is a continuation of the three earlier ones in this series on the application of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates (Harvey, 1999 (Harvey, , 2006 (Harvey, , 2008 and brings the coverage of the literature to the end of 2004. The review is intended to illustrate both developments in technology and in the way in which analysis of carbohydrates contributes to scientific knowledge in general.

Matrix-assisted laser desorption/ionization (MALDI) continues to be a major technique for the analysis of carbohydrates although electrospray, particularly with quadrupole-time-offlight (Q-TOF) instruments, is becoming increasingly popular. Advantages of the MALDI-Q-TOF combination for proteomics and glycomics have been stressed in a brief focus paper (Huang, 2003) . Figure 1 shows the year-by-year increase in papers reporting use of MALDI MS for carbohydrate analysis for the period 1990-2004. As the review is designed to complement the earlier work, structural formulae, etc. that were presented earlier are not repeated. However, a citation to the structure in the earlier works is indicated by its number with a prefix (i.e., 1/x refers to structure x in the first review and 2/x to the second). Other reviews and review-type articles directly concerned with, or including MALDI analysis of glycoconjugates that have been published during the review period include general reviews by Harvey (2003) , Stuhler and Meyer (2004) , and Zaia (2004) , a review of capillary electrophoresis-mass spectrometry for glycoscreening (Zamfir & Peter-Katalinic, 2004 ) and a review on new methods for mass spectrometry of bioorganic macromolecules (Cristoni & Bernsrdi, 2003) . More specific reviews include those on the glycosylation of Caenorhabditis elegans , characterization of substituent distribution in starch and cellulose derivatives (Richardson & Gorton, 2003) , derivatization of carbohydrates for chromatographic, electrophoretic, and mass spectral structural analysis (Lamari, Kuhn, & Karamanos, 2003) , structure of bacterial lipopolysaccharides (Caroff & Karibian, 2003) , analysis of post-translational modifications (Cantin & Yates, 2004; Jensen, 2004; Seo & Lee, 2004) , the use of MALDI MS to detect enantioselectivity in gas-phase ion-molecule reactions with carbohydrates such as cyclodextrins (Speranza, 2004) , synthesis of heparan and heparin sulfate fragments , analysis of protein glycation products (Horvat & Jakas, 2004; Kislinger, Humeny, & Pischetsrieder, 2004) , carbohydrate biosensors (Jelinek & Kolusheva, 2004) , synthesis and discovery of oligosaccharides and glycoconjugates for the treatment of disease (Macmillan & Daines, 2003) , dendrimers in drug research (Boas & Heegaard, 2004) , combinatorial carbohydrate synthesis (Baytas & Linhardt, 2004) , chemical tagging strategies for proteome analysis (Leitner & Lindner, 2004) , capillary electrophoresis of biopharmaceutical products (Kakehi, Kinoshita, & Nakano, 2002) and the use of mass spectrometry to study congenital disorders of glycosylation type IIx (Mills et al., 2003b) .

A comparison between spectra obtained with a conventional quadrupole ion trap (QIT) (electrospray ionization) and a MALDI-QIT-reflectron-TOF instrument has shown that the latter instrument produces significant amounts of metastable fragmentation due to the longer (msec) time span of the QIT trapping process, an observation also made by Harvey et al. (2004) with released N-glycans. Glycopeptides were completely desialylated in the process. Fragmentation (MS 2 ) of glycopeptides produced fragments exclusively from the carbohydrate with the ESI-QIT instrument with peptide fragmentation occurring in MS 3 , whereas the MALDI-QIT-reflectron-TOF instrument yielded both types of fragmentation at the MS 2 stage (Demelbauer et al., 2004) .

Coupling of both vacuum and atmospheric pressure MALDI (AP-MALDI) ion sources with ion-trap instruments has been reviewed (Moyer et al., 2003) and the latter technique investigated as a method for obtaining fragmentation spectra from carbohydrates. High-pressure ion sources have been developed to cool ions in an attempt to reduce or eliminate fragmentation occurring either in-source or post-source. Such fragmentation is the primary factor limiting the use of vacuum MALDI for analysis of acidic glycans and can be considerably reduced with a high-pressure ion source but at the cost of diminished sensitivity. Arabinosazone (1/40) has been used as the matrix for maltohexaose (a-D-Glcp-(a-D-Glcp-) 4 -(1 ! 4)-a-D-Glcp, 1) in positive ion mode ([M þ Na] þ ion) and for 6 0 -sialyllactose (2) in negative mode ([MÀH] À ). The spectrum of the latter compound contained two abundant fragments, an O,2 A 3 and a C-ion (Domon and Costello (1988) nomenclature, see first review, Scheme 3) whereas the major glycosidic fragments in the positive ion spectrum were produced by B or Y cleavages. 

Because of the near physiological conditions attainable at atmospheric pressure with glycerol as the matrix, it has been possible to observe non-covalent complexes between carbohydrates and synthetic peptides that were designed to mimic the binding sites of three members of the siglec family. 3 0 -(3) and 6 0sialyllactose (2), were used as the carbohydrate ligands with 3sialyllactose maintaining its binding specificity for sialoadhesin (Von Seggern & Cotter, 2004) . A new AP-MALDI technique named atmospheric pressure infrared ionization from solution (AP-IRIS) that is claimed to be 16 times a sensitive as AP UV-MALDI has been implemented on a QIT mass spectrometer . The method operated in the absence of an extraction Year 2004 Year 2002 Year 2000 Year 1998 Year 1996 Year 1994 Year 1992 Year 1990 Number of publications 0 100 200 300 400 500 FIGURE 1. Yearly totals of the number of publications containing work relating to the application of MALDI mass spectrometry to carbohydrates and glycoconjugates. field and used a high power IR laser with aqueous glycerol as the matrix. Spectra consisting of [M þ Na] þ ions were obtained from several high-mannose and complex N-linked glycans with much better signal/noise ratios than with corresponding amounts ionized by UV-MALDI from 2,4,6-trihydroxyacetophenone (THAP, 1/44 

Matrix-assisted laser desorption/ionization (MALDI) plates sprayed with the hydrophobic 3M product, ScotchGard TM , a water repellent material for coating fabrics, has the effect of reducing the MALDI spot size and improving sensitivity (Owen et al., 2003) . For cleaning, the coating could easily be removed with methyl-t-butyl ether and the film reapplied to the clean plate. Experiments with the carbohydrate-containing antibiotic, erythromycin A (4), showed increases in sensitivity of two-to threefold when ionized from the coated targets compared with signals from uncoated plates. 

A microdeposition device has been constructed which mixes effluent from capillary electrochromatography or microcolumn liquid chromatography with the MALDI matrix and deposits the mixture onto the MALDI plate. Dextrin and N-linked glycans from ribonuclease B were successfully analyzed (Tegeler et al., 2004 ). An array of 96 perforated nanovials manufactured by silicon microfabrication and filled with 40 nL of reversed-phase beads has been developed as a MALDI target for peptide analysis. It was successfully used to examine peptides and glycopeptides from prostate-specific antigen (Ekström et al., 2004) .

Liquid chromatography-mass spectrometry (LC-MS) has the disadvantage that the entire sample is consumed after exiting the HPLC column. Lochnit and Geyer (2004) have used nano-LC-MALDI-TOF MS to examine tryptic peptides and glycopeptides, a technique that allows more detailed MS investigation of the effluent to be made. The technique also produced enhanced detection of glycopeptides in the presence of peptides and improved characterization of the attached glycans by optimizing the experimental conditions.

Investigations into the mechanism of proton transfer from dihydroxybenzoic acid (DHB) isomers continues. Yassin and Marynick (2003) have calculated the gas-phase acidities of the radical cations of all six dihydroxybenzoic acid (DHB) isomers using density functional theory and found excellent agreement with experimental measurements. The 2,5-isomer (1/26), the most effective matrix, was the least acidic (in this review the abbreviation DHB is used for the 2,5-isomer, unless stated otherwise). The results indicate that, as proposed earlier (Gimon et al., 1992) , deprotonation occurs at the phenolic rather than the acidic site as the result of resonance stabilization of the resulting ion. This conclusion is consistent with the theory proposed by Harvey (1993) that one of the reasons that the 2,5-isomer is so effective a matrix is that it is the only one that can form a p-benzoquinone-type (5) structure after photochemical decarboxylation.

Sugars, however generally ionize as [M þ Na] þ ions for which the factors governing formation are much less clear. A recent study by Antonopoulos et al. (2003) on the mechanism of attachment of sodium to glucose and its methylated derivatives found no simple correlation between ion yield and factors such as volatility and hydrophobicity. A study by Luxembourg et al. (2003) using the high resolution capability of matrix-assisted secondary ion mass spectrometry has demonstrated crystal inhomogeneity among DHB crystals on MALDI targets. Some crystals were free of sodium whereas others contained sodium and sample. Yet other areas of the target contained mostly sodium and little matrix. The results were thought to explain the ANALYSIS OF CARBOHYDRATES AND GLYCOCONJUGATES & phenomenon of ''sweet spots'' frequently observed with MALDI targets.

Proton-affinity values for 15 common matrices have been measured by a kinetic method. a-Cyano-4-hydroxycinnamic acid (CHCA, 1/23) and DHB showed the lowest affinity whereas 2-(4 0 -hydroxyphenyl)azobenzoic acid (HABA, 1/32) and norharmane (1/35) had the highest affinity. In general, the rank order was similar to that found by other investigators. With bcyclodextrin (6, cyclic-(1 ! 4)-a-D-glucose) 7 as the reference compound in negative ion mode, matrices with low proton affinity, such as DHB (204.4 AE 0.17), produced no [MÀH] À ions whereas those with high affinity, such as nor-harmane (233.0 AE 0.44), did (Mirza, Raju, & Vairamani, 2004b) . Gasphase potassium binding energies of several MALDI matrices have also been published .

Cyclic-((1→4)-α-D-Glc) 7 6. β-Cyclodextrin Fournier et al. (2003) have measured the ablation volume of DHB crystals under different laser powers and found that, although the ablation volume increased only slowly with increasing laser power, the signal rose dramatically. They interpreted their results as involving a very rapid ablation to produce a plume that was further ionized by the incoming laser beam. Frankevich et al. (2003) have discussed the role of photoelectrons on MALDI sensitivity. Suppression of photoelectrons with a film of insulating material on the target; the authors used Scotch Magic Tape; was found to produce an increase of up to two orders of magnitude in sensitivity and an improvement in resolution. Bashir, Mutter, and Derrick (2003b) have investigated long-chain esters of DHB as matrices and found some improvement in performance, particularly after the addition of iodine to those compounds that contained a double bond in the chain. The mechanism producing this enhancement in performance by the addition of iodine was unclear but the reason for the improved performance with some of the long-chain compounds was thought to be the formation of micelles that enhanced the matrixanalyte interactions.

5-Amino-2-mercapto-1,3,4-thiadiazole (AMT, 7) has been described as a new matrix for carbohydrates (Mirza et al., 2004a) . Its performance was similar to that of DHB although slightly higher-mass compounds could be ionized from dextran-5000. Kéki et al. (2004) have ionized simple organic molecules such as a peracetylated isoflavone glycoside (8) from common matrices such as DHB doped with silver trifluoroacetate to give [M þ Ag 3 ] þ species. These ions were shown to be more stable than the corresponding [M þ Ag] þ ions. The latter ions and their Cu þ counterparts, fragmented in a similar manner to those from Na þ and Li þ (Kéki et al., 2004) . Carbon nanotubes, prepared from coal by an arc discharge have been reported as a novel matrix for low molecular weight peptides and b-cyclodextrin . Spectra showed a very low background as the result of the absence of matrix peaks and ions produced by fragmentation. To obtain the spectrum, a well-dispersed ethanolic solution of nanotubes was deposited onto the MALDI target and allowed to dry. The sample solution was then deposited onto the nanotubes and dried with hot air. Two papers reporting the use of fine cobalt powder for the analysis of lepidimoic acid (9) from okra pectic polysaccharide (Hirose et al., 2003) and of lepidimoide (10) from okra mucilage have appeared (Hirose, Endo, & Hasegawa, 2004) . 

A mixture of DHB and CHCA has been shown to give enhanced performance for the ionization of glycopeptides than use of the individual matrices. The mixed matrix showed better tolerance towards the presence of salts and impurities and improved peptide sequence coverage (Laugesen & Roepstorff, 2003) . A thin layer of very homogeneous crystals has been produced by mixing THAP with nitrocellulose and used to analyze pectin digests (Mohamed, Christensen, & Mikkelsen, 2003) .

Atmospheric pressure MALDI (AP-MALDI) has the advantage over vacuum MALDI of collisional stabilization of fragile molecules such as carbohydrates containing sialic acid residues. The technique is also compatible with the use of liquid matrices whose volatility presents problems under vacuum conditions. Von Seggern, Moyer, and Cotter (2003a) have compared water, glycerol, and nitrobenzyl alcohol (1/21) for ionization of sialylated glycans with an IR laser (Er:YAG, 2,490 nm) and have found that glycerol provides the longest lasting signal with the best signal/noise ratio. Water was too volatile to survive for long under the heat produced by the laser. Intact sialylated glycans were desorbed as [MÀH] À and [M þ Na] þ ions under negative and positive conditions respectively. In a further study (Von Seggern, Zarek, & Cotter, 2003b) , the glycerol matrix was doped with alkali (Li, Na, K) alkaline earth (Ca) or transition metal cations (Ca, Co) for production of adducts containing one or two singly charged cation or one doubly charged cation. Adduction proved to be efficient with lithium or potassium being able to replace the more common sodium adduction when salts (Cl À or I À ) containing these metals were used as the doping agents. Cobalt adduction was achieved by use of cobalt powder added to the glycerol. Fragmentation (see below) was obtained with an ion-trap instrument. The use of glycerol as the matrix with an IR laser under atmospheric pressure conditions has also enabled non-covalent sugar-sugar complexes to be observed (Von Seggern & Cotter, 2003) . The stability of the complexes varied with different sugars potentially due to the strengths of the hydrogen bonding networks. Fragmentation of the complexes was also a function of structure; some complexes, particularly those containing sialic acid, fragmented by loss of the acid before breakdown of the complex whereas complexes between neutral molecules tended preferentially to dissociate into monomer units.

Ionic liquid matrices appear to show great promise for carbohydrate analysis. The matrix DHB-butylamine (DHBB) was reported to give improved shot-shot reproducibility compared with solid matrices such as DHB, and reduced the relative standard deviation by 50%. It also produced much less fragmentation from compounds such as sialylated carbohydrates (Mank, Stahl, & Boehm, 2004) . Greatly improved quantitative performance has also been reported by Zabet-Moghaddam, Heinzle, and Tholey (2004) .

Derivatization techniques for analysis of carbohydrates have been reviewed (Gao et al., 2003; Suzuki et al., 2003b) .

Advantages of analytical derivatization have been emphasized by Rosenfeld (2003) with, among others, examples from the reductive amination of carbohydrates for improving the interpretation of fragmentation spectra and for modifying behavior in chromatographic systems. MALDI is frequently used to monitor reducing-terminal labeling, used to attach fluorophores or chromophores to carbohydrates for chromatographic detection as illustrated in a recent study of glycans from the Tamm-Horsfall glycoprotein (Rohfritsch et al., 2004) and the use of 2-AAderivatives to monitor glycosylation of therapeutic glycoproteins (Dhume et al., 2002) . Benzylamine (2/9) derivatization was used by mainly for ESI-MS/MS analysis of small and N-linked glycans. However, exoglycosidase sequencing was performed on a MALDI target and it was observed that the derivative did not inhibit the enzymic reaction. An, Franz, and Lebrilla (2003a) have derivatized several sugars, including N-linked glycans released from ribonuclease B, with benzylamine and quaternized the product with methyl iodide as originally performed by Broberg, Broberg, and Duus (2000) but with a procedure not involving the use of resins. The product allowed separation of isomers by capillary electrophoresis (CE) and also gave strong MALDI spectra on account of the inbuilt charge.

It is possible to remove labels attached by reductive amination; thus, 2-aminopyridine (2-AP, 1/52)-derivatized Nglycans have been underivatized by a process that involved catalytic hydrogenation and hydrazinolysis using the method reported by Hase (1992) to give the reducing-terminal amine which was converted to an aldehyde using the Sommlet reaction (hexamethylenetetramine and acetic acid) (Takahashi, Nakakita, & Hase, 2003a ).

Phenylhydrazine (11) has been used to form hydrazone derivatives of N-linked glycans for both MALDI and electrospray ionization. Because the derivatives were not reduced, as with reductive amination, no post-derivatization clean-up was necessary. Derivatives could be prepared on-target by addition of a solution (0.5 mL), prepared by dissolving phenylhydrazine (1 mL) in 10 mL of water/methanol (4:1 v/v), directly to the glycanmatrix mixture on the MALDI target and allowing the mixture to dry under ambient conditions for 45 min. No osazone formation was reported and the derivatives produced [M þ Na] þ ions. Postsource decay (PSD) spectra were reported to be simpler than those of the underivatized glycans and to be dominated by B/Y internal fragments (Lattova & Perreault, 2003a,b) . HN NH 2

Several hydrazones containing either a constitutive cationic charge, such as those formed from Girard's T reagent (1/55, quaternary ammonium) or from hydrazines carrying a guanidine group (e.g., 12), have been shown to produce increases in sensitivity and to suppress signals from peptides, thus allowing N-linked glycans to be detected in the presence or tryptic peptides without extensive clean-up . Differences in sensitivity among the derivatives were observed. Among those containing a charge, and which formed M þ ions, the pyridinium ANALYSIS OF CARBOHYDRATES AND GLYCOCONJUGATES & derivative (13) was the most efficient, producing a detection limit of 1 fmol. The guanidine derivatives formed [M þ H] þ ions and some produced equal detection limits. It would appear that the most efficient derivatives possessed both good ion-forming properties and high lipophilicity. Improved detection limits have also been achieved by use of hydrazide derivatives of the cyanine dyes or Cy-5 (15) that contain a constituent positive charge . N-glycans from chicken ovalbumin could be detected with near independence from the type of matrix. PSD spectra contained mainly Y-type ions consistent with localization of the positive charge on the derivative. 

Sialic acids can be stabilized for MALDI analysis by formation of methyl esters to remove the very labile acidic proton on their carboxyl groups. Migration of this hydrogen atom is largely responsible for lability of this carbohydrate. A recent example of methyl ester formation is in a study of the N-linked glycosylation of the glycoprotein, N-cadherin, from human melanoma cell lines (Ciolczyk-Wierzbicka et al., 2004) . The acids were first converted into their sodium salts with a Dowex AG50 resin (Na þ form) and these salts were then reacted with methyl iodide. Other examples are included below.

Removal of contaminants such as salts, buffers, etc. is essential to obtain high quality spectra. A variety of methods are used, the majority being dialysis, use of various membranes such as Nafion 117 (Börnsen, Mohr, & Widmer, 1995) or various resins. Methods for preparing samples for mass spectrometric analysis have been reviewed (Perreault, 2004) . Some examples of those during the review period are listed below.

Drop dialysis has been used in clean-up of lipooligosaccharide (LOS) from Moraxelle catarrhalis by Luke et al. (2003) and a Nafion membrane for on-probe clean-up of O-acetylated glucomannans from birch and aspen (Teleman et al., 2003) .

Dowex AG50 resins in various forms have been used for analysis of N-glycans from the moss Physcomitrella patens (Viëtor et al., 2003) (AG 50W-X2 form), deacylated lipopolysaccharide (LPS) from Vibrio parahaemolyticus strains (Hashii et al., 2003a,b) (50 W Â 8, H þ form to remove cations) and extracellular polysaccharide (EPS) from Burkholderia cepacia (Cescutti et al., 2003) (50 W Â 8-200) . AG50 combined with AG3 (anions) in a gel loader tip have been used for N-linked glycans (Hoja-Lukowicz et al., 2000; Ciolczyk-Wierzbicka et al., 2004) . Porous graphitized carbon has been employed to desalt quaternized benzylamine derivatives (An, Franz, & Lebrilla, 2003a) and Hypercarb columns have been used by Mills et al. (2003b) for N-linked glycans released from human plasma glycoproteins proteins and by Forno et al. (2004) (Higai et al., 2003a) and sialyl Lewis X antigen-expressing glycoproteins secreted by human hepatoma cell line (Higai et al., 2003b) and Wada, Tajiri, and Yoshida (2004) have employed both cellulose or Sepharose for the separation of tryptic glycopeptides from tryptic peptides. C18-solid-phase extraction followed by Carbograph column was used by Ohl et al. (2003) to remove detergents from Nglycans. Salts, etc. ware eluted from the columns with water, neutral glycans with 25% MeCN and acidic glycans with 25% MeCN/0.05% trifluoroacetic acid (TFA). Amberlite IR-120 (H þ ) was used by Frirdich et al. (2003) to purify core oligosaccharide from E. coli. Release of N-glycans with protein-N-glycosidase F (PNGase F) is more efficient if the glycoprotein is pre-digested with trypsin or other protease. However, the resulting peptides can interfere with subsequent analysis and are difficult to remove with octadecyl (C 18 ) silica cartridges, cation exchange columns or graphitized carbon cartridges. To overcome this problem, Nakano, Kakehi, and Lee (2003b) have modified the amino groups of the peptides with 2,4,6-trinitrobenzene-1-sulfonate (16) to render them more hydrophobic so that they can be more strongly retained on C 18 or graphitized carbon.

16. 2,4,6-trinitrobenzene-1-sulfonate

Affinity capture of glycoproteins to a MALDI target has been reported by Koopmann and Blackburn (2003) . The target was coated with poly-L-lysine poly(ethylene glycol)-biotin polymer followed by tetrameric neutravidin. This surface then acted as a capturing surface for biotinylated proteins which could be adsorbed and desalted by washing. MALDI spectra were obtained following addition of CHCA to the surface. Proteinprotein interactions could also be studied with this treated target and, furthermore, it could be used to isolate glycoproteins by lectin binding. Thus, biotinylated wheat germ agglutinin bound to the surface was used to further capture fetuin. The resulting MALDI spectrum contained ions from the biotinylated lectin, neutravidin and fetuin.

Glycopeptides, such as those obtained from protease digests, frequently give weak signals in the presence of hydrophobic peptides. A simple method for separating hydrophobic from hydrophilic peptides on a MALDI target has recently been devised (Kjellström & Jensen, 2003 ). An aqueous solution of the peptide mixture was placed on the target followed by an immiscible solvent, such as ethyl acetate with or without added matrix. The organic solvent extracted the hydrophobic peptides and was allowed to evaporate, after which, the residual aqueous phase was transferred to another part of the target and allowed to dry after addition of DHB. MALDI spectra could then be acquired from both the hydrophobic and hydrophilic fractions.

A paper on quantitation of glucose (1/4) in a study on enzyme kinetics has highlighted problems in the use of MALDI-MS for quantitative work and how the technique can produce good quantitative results in spite of the popular belief that the technique is not quantitative. The inhomogeneity of the target and variations in the shot-to-shot laser intensity can be compensated for by averaging many shots from different target positions. It is also important to eliminate any spectrum in which the A/D converter is saturated. By use of a [U-13 C]-labeled internal standard and measurement of the [M þ Na] þ ions, a linear calibration curve was obtained with a correlation coefficient of r ¼ 0.991. The mean standard deviation was 6% but the authors (Bungert, Heinzle, & Tholey, 2004b) state that this could be improved if more spectra were averaged although this would have increased the analysis time.

A MALDI method for the quantification of glucose from hydrolyzed starch has been reported. Sorbitol (1/42) was used as the internal standard and DHB provided the matrix even though it produced peaks in the same region, but not at the same mass as the analytes. Calibration curves were linear over the range 0.1-10 pmol added to the target and the method compared favorably with the normal colorimetric method for measuring glucose. Even though the MALDI results showed a greater degree of variability, this was more than compensated for by the speed of analysis (Grant et al., 2003) .

A quantitative MALDI-TOF MS method as also been developed for screening of ten pyranose oxidase variants. The isotopic labeled internal standard, [U-13 C]-glucose and the ionic liquid matrix, DHB/pyridine, were used with aliquots of enzyme reaction mixtures without pre-purification steps (Bungert et al., 2004a) . The ionic, liquid matrix enabled spectra to be recorded from most areas of the target (only 7 out of 200 spots failed to produce a signal). The mean standard deviation for glucosone was 11.8% and the results were in good agreement with HPLC measurements.

Glycosidic and cross-ring cleavages produce most of the fragment ions from carbohydrates. A third type of fragmentation mechanism has now been reported involving hexacyclic hydrogen rearrangements. The products were proposed to consist of unsaturated sugar rings lacking two oxygen atoms as shown in Scheme 1 (Spina et al., 2004) .

Post-source decay (PSD) fragmentation of per-acylated isoflavone glycosides (e.g., 8) cationized with various metals and hydrogen have shown that the number of fragment ions decreased in the order Li þ =Na þ > Ag þ > Cu þ > H þ > K þ > Rb þ =Cs þ , roughly in line with earlier studies of underivatized carbohydrates (Kéki et al., 2003) . Fragments were largely glucosidic and losses of acetic acid and ketene but, unusually, there were a few fragments in some of the metal-cationized spectra that had lost the metal. Loss of acetic acid and ketene was highest for the lighter metals. The authors attributed this observation to elimination of metal acetates.

Neutral and some acidic carbohydrates have been shown to form stable adducts with chlorine that survive the MALDI process to give [M þ Cl] À ions from matrices such as harmine (1/36) that have gas-phase acidities lower than or close to that of HCl (Cai, Jiang, & Cole, 2003) . Such adducts open the way for the production of negative ion fragmentation spectra from carbohydrates that, in many cases, produce more informative spectra than their positive ion counterparts. In the above publication, the adduct of the disaccharide D-turanose (a-D-Glcp-(1 ! 3)-D-Fru, 17) was reported to fragment initially by loss of HCl to give a PSD spectrum containing both glycosidic and MS/MS analysis of N-acetyllactosamine-6,6 0 -disulfate (18) and its 2 0 -epimer produce different fragmentation patterns under several mass spectrometric methods (fast-atom bombardment (FAB), ESI and MALDI-PSD). The PSD spectra were characterized by an abundant ion at m/z 431 that was produced from the 2a-epimer by elimination of both the N-acetylamino-group and the 6-sulfate (Ohashi et al., 2004) . Muzikar et al. (2004) have found that many carbohydrates containing an amino group at the reducing terminus show enhanced PSD fragmentation with production of more abundant cross-ring fragments. N-linked glycans are normally released enzymatically in this form but cross-ring fragmentation of these branched compounds was not enhanced as much as that from linear compounds and was inferior to the high-energy fragmentation obtained with a TOF/ TOF mass spectrometer. 

A novel method of ion isolation for CID has utilized IR-induced fragmentation and consequent removal of unwanted ions by use of a Nd:YAG laser that shone directly into the analytical cell of a modified Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer (Xie, Schubothe, & Lebrilla, 2003) . The method differed from existing techniques in that it did not involve diverting the unwanted ions with magnetic or electric fields. The paper reported the isolation of g-cyclodextrin (1/65) from a mixture containing maltotetraose ((b-(1 ! 4)-linked D-glucose) 4 , 19) and maltohexaose both of which also produced fragments in the analytical cell prior to irradiation with the IR laser. Infrared multiphoton dissociation (IRMPD) of alkali metaladducted carbohydrates has also been studied in this instrument and compared with CID. Although higher energy fragmentation could be obtained with CID, IRMPD provided continuous energy transfer to the fragment ions with the result that more extensive fragmentation was achieved .

Fragmentation of maltohexaose and high-mannose glycans (positive ion) with a TOF-TOF mass spectrometer with air as the collision gas has been shown to give high-energy-type fragmentation with the production of abundant cross-ring cleavage ions and, in particular, X-type cleavages not seen in the low energy spectra. Isomers of Man 7 GlcNAc 2 (20) could be distinguished with respect to the antenna (3 or 6) to which the seventh mannose was attached by the O,4 A 3 and 3,5 A 3 ions (Mechref, Novotny, & Krishnan, 2003) . Similar results have been reported by with a series of smaller sugars. A study of the effect of DHB or CHCA on fragmentation patterns of native and permethylated oligosaccharides in a TOF/TOF mass spectrometer has shown that, whereas CHCA promoted mainly glycosidic cleavages, DHB initiated glycosidic, cross-ring and internal cleavages with X-type cross-ring cleavages among the most abundant ions. Permethylation produced increased sensitivity and spectra that were easier to interpret. Studies with avidin from a DHB matrix allowed most of its N-glycans to be identified (Stephens et al., 2004a) . A comparison of matrices used for ionization of 2-AP-labeled complex N-linked glycans with a TOF-TOF mass spectrometer has shown that, whereas CHCA produced only sodiated fragments in MS 2 spectra from [M þ Na] þ parent ions, DHB produced both sodiated and protonated ions. It was suggested that the two matrices produced parent ions in different excited states . However, CHCA was more effective than DHB in producing abundant PSD ions. Although providing additional information through the production of more cross-ring cleavages, acidic glycans showed extensive fragmentation before reaching the collision cell. Sialylated glycans, however, can be stabilized by permethylation or methyl ester formation, as discussed above.

Negative ion fragmentation appears to be gaining more in popularity because of its ability to produce abundant cross-ring cleavages. The [MÀH] À ion generated by AP-MALDI from 6 0sialyl-lactose (6 0 -SL, 1) using a glycerol matrix, for example, yields the O,2 A 3 ion as the most abundant fragment (Von Seggern, Moyer, & Cotter, 2003a) . C-ions were prominent in the spectrum of this and larger sugars such as LSTa (Neu5Ac-a- Glc, 22) and the spectra easily allowed the location of sialic acids to be determined. Cross-ring and C-type fragments were also prominent in the spectra of lithiated adducts and, in particular, the spectra of lithium salts of sialic acids (Von Seggern, Zarek, & Cotter, 2003b) . Fragmentation of the [M þ Li] þ and [M þ Co] þ ions showed no significant difference between 3 0 -(3) and 6 0 -SL (2) but the spectra of the [MÀH þ 2Li] þ ions were different. In general, replacing the acidic proton of the sialic acid group by salt formation prevented ready loss of sialic acid under positive ion conditions and accentuated differences between the spectra of isomeric sugars containing sialic acid at different positions.

Neu5Ac-α-(2→3)-Gal-β-(1→3)-GlcNAc-β-(1→3)-Gal-β-(1→4)-Glc 22. LSTb Gal-β-(1→3)-GlcNAc-β-(1→3)-Gal-β-(1→4)-Glc | Neu5Ac-α-(2→3)

Studies with a MALDI-ion-trap-TOF instrument have shown that many of the fragments in the positive ion spectra of N-glycans originate from multiple sites. In the spectra of high-mannose glycans an ion formed by loss of the chitobiose core and 3antenna is diagnostic for the composition of the 6-antenna. However, in the spectra of biantennary glycans (23), the abundant ion at the equivalent mass (m/z 712, Hex 3 -HexNAc) was shown to be formed predominantly from the B 5 ion (loss of the reducingterminal GlcNAc) and Gal-GlcNAc from each antenna .

Symbols as for 20 plus % = galactose

The programming language Java has been used to write a program for identifying the total monosaccharides present in a tryptic glycopeptide containing up to six O-linked glycans from the hinge region of human serum immunoglobulin 1 (IgG1). The program also calculated the amount of each glycopeptide as a percentage of total peak area .

A modification and extension to the StrOligo algorithm (Ethier et al., 2002) for assigning structures of N-linked glycans from their CID spectra has been published (Ethier et al., 2003) . Spectra were recorded with a MALDI-Q-TOF instrument, isotope-stripped and examined for peaks corresponding to monosaccharide losses. The program then built a relationship tree that was analyzed with respect to fragment ion types and adduct. [M þ Na] þ , the ions usually encountered in MALDI spectra of carbohydrates are now catered for as well as the [M þ H] þ ions featured in the original version of the program. Negative ion fragmentation was also accommodated. Greater attention to known biochemistry was incorporated in the new algorithm and the program was able to annotate spectra with the most probable structure.

A web-based tool entitled GLYCO-FRAGMENT, to be found at http://www.dkfz.de/spec/projekte/fragments/ accepts carbohydrate structures in the extended IUPAC nomenclature (http://www.chem.qmw.ac.uk/lupac/2carb/38.html), as developed for the carbohydrate database, CarbBank, and calculates the masses of all possible fragments. The presence of reducingterminal derivatives, various adducts such as sodium or lithium, persubstituted (e.g., permethyl) derivatives and substituents such as sulfate, acetate and phosphate are also accepted. B-and Y-type fragments are listed by default and masses of C-, Z-and crossring fragments can be obtained on demand. Internal fragments are not catered for. Output is in the form of a list or, in ''view as structure'' mode, ions associated with cleavage of each bond are shown when the user moves a cursor over a specific glycosidic linkage (Lohmann & von der Lieth, 2003) . GlycoSearchMS (http://www.dkfz.de/spec/glycosciences.de/sweetdb/ms/) compares each peak in a mass spectrum with calculated fragments from all structures in the SweetDB database and the best matches are displayed (Lohmann & von der Lieth, 2004) . Constituent monosaccharides can be obtained from the program GlycoMod (http://www.expasy.ch) as demonstrated by Ma et al. (2003a) to assign compositions to N-glycans released from the glycoprotein rat selenoprotein P. Reviews of available databases relating to glycomics have been published (von der Lieth, 2004a,b) .

Work in this area is summarized in Table 1 . A comparison of the behavior of dextrans in positive and negative ion MALDI has shown the expected formation of [M þ Na] þ ions in positive mode whereas the negative ion spectra contained ions at [M-1-120] À as the result of fragmentation. Under ESI conditions on a Shimadzu-Kratos SEQ instrument, positive ionization again produced [M þ Na] þ ions but with a bias towards the lower masses. The negative ion spectra were dominated by extensive A-type fragment ions. MALDI was reported to be more sensitive than ESI but ESI was more useful for structural studies (Cmelík, Stikarovská, & Chmelík, 2004) . Comparisons of fragmentation modes for the [M þ Na] þ ions from these compounds have shown that, whereas PSD, ISD, and CID all produced glycosidic cleavages, only ISD and CID produced significant cross-ring cleavage. Localization of the charge to the reducing terminus by formation of a 1-phenyl-3methyl-5-pyrazolone derivative (see 1/61) restricted cleavage to the non-reducing terminus (Bashir et al., 2004) .

The b-D-fructan (inulin; for fructose, see 1/16) from dahlia tubers and glucose syrup from potatoes, together with similar polysaccharides from red onion and Jerusalem artichokes, were used as reference compounds by Stikarovská and Chmelík (2004) to evaluate the best matrices for these compounds. Linear-MALDI-TOF analysis gave stronger signals than reflectron-TOF; DHB was found to be the best matrix for starch and THAP for inulin. Less effective matrices were CHCA, HABA, 3aminoquinoline (3-AQ, 1/24) and sinapinic acid (1/48). Depolymerization of most of these very large molecules is necessary before MALDI analysis. Enzymatic treatment is usually used (details in Table 1 ) but heat treatment is also common. Acetylation of glucuronoxylans and glucomannans has received considerable attention (Table 1) with some evidence of acetyl group migration during depolymerization (Kabel et al., 2003) . Acetylated glucuronoxylans and glucomannans have been recovered from flax shive following hydrothermal treatment but in this case, acetyl migration was not reported . Acetylation has also been found on the mannose residues of glucomannans from birch and aspen wood and appear to be randomly distributed (Teleman et al., 2003) . MALDI-TOF spectra from these two papers consisted of an extensive series of peaks with a mass separation of 42 units (masses up to 5,500) but considerable simplification was found after deacylation. Fernández et al. (2003) have examined arabinoxylans from wheat by MALDI and ESI mass spectrometry and detected structures with up to 22 monosaccharide residues. As arabinose (1/2) and xylose (1/3) are isobaric, it was not possible to study the distribution of arabinose residues along the xylose backbone. However, this problem was overcome by permethylation; arabinose residues attracted three methyl groups, unsubstituted internal xylose residues, two and substituted residues one group.

Matrix-assisted laser desorption/ionization (MALDI) conditions for determination of the substitution patterns in methyl- (Momcilovic et al., 2003b) and carboxymethyl-cellulose (Momcilovic et al., 2003a) have been evaluated. Depolymerization was achieved both by use of enzymes or acid and the products fractionated by size-exclusion chromatography (SEC). DHB, CHCA, HABA and indoleacrylic acid (IAA, 3/5) were tested as matrices for methylcellulose but little difference was found between them. The choice of solvent, however, did have a significant effect, probably due to differential solubility of the variously methylated compounds. Measurements on two acid-depolymerized samples of methylcellulose with different degrees of substitution gave values that agreed well with the involved in 2 ary cell wall metab. (Goujon, et al., 2003) Argania spinosa (Argan tree) Identification of novel XUFG motif Aspergillus wentii Araf, Gal Structure of oligosaccharides that modulate intestinal immune system (Taguchi, et al., 2004) Azorhizobium caulinodans Glc, Man Random acetylation on Man residues of glucomannans. (Teleman, et al., 2003) Barley Starch Enzyme L-TOF (+ve) (DHB) Glc Quantification of glucose from starch. Sorbitol as internal stand. (Grant, et al., 2003) Barley (hull-less)

Heat MALDI Glc Study of structural changes caused by heating (Li, et al., 2004c) Birch wood Glucomannan -TOF (DHB) SEC, HPAEC, NMR Glc, Man Random acetylation on Man residues of glucomannans. (Teleman, et al., 2003) Brassica campestris L.

(Field mustard)

Endo-(1→4)-βxylanase TOF(DHB) GLC, GC/MS, SEC Rha, Fuc, Ara, Xyl, Man, Gal, Glc Structural characterization of polysaccharides from seed cake (Ghosh, et al., 2004) Chaetosartorya chrysella (Rosenbohm, et al., 2003) Coffea arabica (green coffee)

Endomannase TOF (DHB/HIQ) Man, Gal, Acetyl groups Structural characterization (Oosterveld, et al., 2004) Commercial sample Structural determination in pharmaceutical formulations (Kühn, et al., 2003) (Continued ) Structural characterization. Inhibition of salivary α-amylase (Kandra, et al., 2004) Corn Starch Enzyme L-TOF (+ve) (DHB) Glc Quantification of glucose from starch.Sorbitol as internal standard. (Grant, et al., 2003) Craterostigma wilmsii

Endo-glucanase R-TOF (DHB) Ara, Rha, Xyl, Man, Gal, Glc, GlcA, GalA Study of cell-wall composition of dehydrated resurrection plant.

More Gal than hydrated leaves. (Vicré, et al., 2004) Eleusine coracana (Kabel, et al., 2003) Flax shive Acetyl-glucomannan Hydrothermal L-TOF (+ve, -ve) (DHB) SEC Glc, Man No acetyl migration after hydrothermal treatment Flax shive Xylooligosaccharides Hydrothermal L-TOF (+ve, -ve) (DHB) SEC Xyl, Me-GlcA Isolation and characterization of water-soluble hemicelluloses Gelidum amausii

To study antioxidative properties (Wang, et al., 2004a) Gramineae (Doco, et al., 2003) Lasallia pustulata

Pustulan Galactomannan Acetolysis R-TOF (DHB) NMR, IR, GLC

Gal, α-Man Main constituents are pustulan, a β-glucan-chitin complex and a β-(1→3)-glucan (Pereyra, et al., 2003) Lycopersicon esculentum (Tomato)

Xyloglucan-specific endoglucanase TOF (DHB/HIQ) NMR α-Ara, β-Ara, Xyl, Glc, Gal Structure of xyloglucan produced by suspension-cultured tomato cells. (Jia, et al., 2003) Manihot esculenta Crantz,

Starch Iso-amylase TOF ("standard matrix") Glc Study of different strains. One has more water-soluble glycans and unusual starch with low branching (Carvalho, et al., 2004) Mesorhizobium haukuii Cyclic-β-glucan (Guérardel, et al., 2003) Nothogenia erinacea Xylan Xylanase R-TOF (DHB) ESI, NMR Xyl Comparison of xylanases from different families (Nerinckx, et al., 2004) Olea europaea (Olive)

Polygalacturonase, arabinofuranosidase MALDI, HPLC Rha, Gal, Fuc, Xyl, Ara, Man, Glc Structural determination related to oil extraction (Vierhuis, et al., 2003) (Reis, et al., 2003) Picea abies (Spruce wood) Galactoglucomannan Hydrothermal L-TOF (+ve, -ve) (DHB) SEC Man, Glc, Gal, Xyl, Ara Effect of temperature and pH for extn. of hemicelluloses. 2% NaOH increased high mass (14 kDa) extn. but removed OAc groups (Lundqvist, et al., 2003) Picea abies (Spruce wood)

Steam SEC-MALDI Mannan and xylan Investigations of compounds that can replace fossil-based products (Palm & Zacchi, 2004) Potato, Sweet potato (Mou, et al., 2004) Red wine Rhamnogalacturonan II Endogalacturonase Acid hydrolysis

β-D-Apif Structural determination of heptasaccharide from red wine rhamnogalacturonan Scheelea phalerata (Palm) Heteroxylan Partial acid hydrolysis TOF (DHB) , NMR, GC/MS GlcpA, α-GalpA, β-Xylp, α-GlcpA, Araf Structural characterization (Simas, et al., 2004) Schizosaccharomyces pombe Glucans NaOH/Zymolyase TOF (CHCA), NMR

Structural characterization (Sugawara, et al., 2004) Solanum tuberosum (potato)

Isoamylase TOF (DHB)

Glc-phosphate Phosphorylation of starch is required for degradation (Ritte, et al., 2004) Sugar beet pulp Acetylated homogalacturonan Endogalacturonase from Fusarium moniliforme TOF (THAP), HPAEC GalA, Me-GalA Products of enzymolysis show decreasing hydrolysis for more highly acetylated compounds (Bonnin, et al., 2003) Sweet potato

Water TOF (DHB) Glc Investigation of hydrothermal conditions for glucose production (Nagamori & Funazukuri, 2004) Tamarind Xyloglucan Pyrococcus furiosus β1→4-endoglucanase TOF (DHB) GLC Xyl, Glc Structural determination.

Fragments with 2-9 residues. (Marry, et al., 2003) Wheat Arabinoxylans Endoxylanase A R-TOF (DHB), ESI, MS/MS

Structures with up to 22 monosaccharide residues detected. (Fernández, et al., 2003) Wood, various species Hemicelluloses -

Xyl, HexA Xylan more abundant in surface layers of pulp fibres (Dahlman, Jacobs & Sjöberg, 2003) (1) Instrument (matrix) other technique, sample (derivative).

data supplied by the manufacturers. However, data from the carboxymethyl-celluloses was less satisfactory even though samples with different degrees of substitution could easily be distinguished from each other. A method has been published for detecting hydroxyethyl starch in urine. The compound is used as a plasma volume expander by athletes and has been banned by the International Olympic Committee. Partial acid hydrolysis of urine was carried out with TFA and the product was examined directly by MALDI-TOF using super-DHB (s-DHB). The method was claimed to provide unambiguous identification in only 90 min (Gallego & Segura, 2004 ).

Of the matrices DHB, nor-harmane (1/35), CHCA, HABA, IAA, and sinapinic acid, DHB and nor-harmane were found by Xiong et al. (2003b) to give the strongest and best resolved spectra from macrocyclic polysaccharides containing from 19 to 25 sugar residues. Sinapinic acid gave a very weak signal and was only recorded in linear mode. The liquid matrix, CHCA/3-AQ/ glycerol also gave a strong and long-lasting signal but with inferior resolution. Addition of alkali metal salts produced the expected adducts.

A glass slide containing a UV absorbing TiO 2 sol-gel film from which imprinted a-cyclodextrin (CD (cyclic-(1 ! 4)-a-Dglucose) 6 , 24) molecules had been removed has been used to selectively adsorb a-CD from a mixture of a-, b-, and g-CD. The a-CD was detected directly by MALDI-MS without the addition of extra matrix .

Cyclic-(1→4)-α-D-Glc) 6

Experiments with a-, b-, and g-cyclodextrins have shown that the larger molecules have greater affinity for larger alkali metals suggesting that the products are inclusion complexes that can be ionized intact. Sinapinic acid was used as the matrix and under these conditions, dextran, a linear polymer, produced only [M þ H] þ ions (Bashir et al., 2003a) . Inclusion complexes with cyclodextrins have recently been used to investigate the composition of humic acids from Antarctica. The structure of humic acids is still largely unknown even though they are widely distributed in nature. Gajdošová et al. (2003) recorded the MALDI spectra of humic acids with and without g-cyclodextrin and found that several constituents appeared to form inclusion complexes. Thus, for example, a shift in the cyclodextrin peak by 66.0 mass units after addition of humic acid from a standard soil sample was interpreted as consistent with inclusion of a cyclopentadiene radical (C 5 H 6 þ ). Results from the soils from Antarctica were similar. MALDI spectra were recorded without a matrix to simplify the spectra. An investigation of the mechanism of the well-known migration of alkyl-silyl groups from the two-to the three position in cyclodextrins has shown that the receiving oxygen is that from the same ring as the migrating silyl group and not to the adjacent ring (Teranishi & Ueno, 2003) .

Matrix-assisted laser desorption/ionization (MALDI) sample preparation protocols for examination of Curdlan ((( ! 3)-b-D-Glc-(1 ! )) n , 25), a polyglucose obtained from the bacterium Alcaligenes faecalis var. myxogenes 10C3, have been reported. The crude sample was separated into a low molecular weight, water-soluble portion and a high-molecular weight, waterinsoluble portion. The low molecular weight portion was examined from DHB containing ammonium fluoride and gave two low-mass (<4 kDa) polysaccharide distributions differing by 16 Da. The high molecular weight, water-insoluble portion was found to produce good signals from a mixture of DHB and 3-AQ in dimethylsulfoxide (DMSO) that was dried on a hot-plate at 708C. Ions with masses of up to 15,000 were observed (Chan & Tang, 2003) . Spectra of extracellular polysaccharides from the colony-forming prymnesiophyte algae Phaeocystis globosa and P. antarctica showed peaks of less than 1,000 Da, some of which had a repeat pattern of 192 Da but the monosaccharide units were not identified (Solomon et al., 2003) .

(→3)-β-D-Glc-(1→)) n Supplementation of a pre-term formula with a mixture of galacto-and fructo-oligosaccharides with a molecular weight range similar to that of carbohydrates found in human milk as determined by MALDI-TOF analysis has been found to have a stimulating effect on the growth of bifidobacteria in the intestine and results in more frequent produced and softer stools. Thus, prebiotic mixtures such as the studied oligosaccharide mixture might help in improving intestinal tolerance to enteral feeding in pre-term infants (Boehm et al., 2003) .

It is unusual for all but the simplest, low mass glycoproteins to produce MALDI-TOF spectra with resolved glycoforms but broad peaks and significant mass differences between observed and calculated protein masses for larger glycoproteins suggest glycosylation. Thus, epoetins (erythropoietins), with several glycosylation sites occupied by sialylated complex glycans, produced only broad peaks with masses in the range of 30-35 kDa and half-height peak widths of up to 5 kDa when examined by MALDI-TOF (Stanley & Poljak, 2003) . Deglycosylation, however, produced sharp peaks from the protein with only minimal tailing. Human follistatin expressed in CHO cells has two N-linked glycosylation sites at Asn-95 and 259. Its MALDI spectrum contained three peaks; the first at m/z 31,525 corresponded to the unglycosylated protein and the other two at m/z 33,804 and 35,600 were produced by unresolved glycopeptides. LC/MS analysis identified nine complex glycans (Hyuga et al., 2004) . Several partially resolved peaks from 52 to 55.5 kDa suggest glycosylation of human b-secretase catalytic domain, confirmed as N-linked by digestion with PNGase F . Reduction in mass of 1.4 kDa of a humanized anti-HBs Fab fragment by treatment with Endo-H gave a mass that was still 3.7 kDa above that of the protein. The authors speculated that the extra mass might indicate O-linked glycosylation but no proof was provided (Ning et al., 2003a) . Similarly, a reduction in the mass of approximately 4.5 kDa of a lysosomal serine carboxypeptidase from Trypanosoma cruzi following Endo-H treatment suggested the presence of two to three N-linked glycans (Parussini et al., 2003) . Other carbohydrates detected by massdifference measurements of proteins before and after deglycosylation include GlcNAc in Cu/Zn-superoxide dismutase from fungus Humicola lutea 103 (Dolashka-Angelova et al., 2004b) and the high-mannose glycan Man 10 GlcNAc 2 in ovotransferrin expressed in Pichia pastoris (Mizutani et al., 2004) .

Matrix-assisted laser desorption/ionization (MALDI) analysis is also useful for detecting the absence (Perteguer et al., 2004) or modification of glycosylation. Thus, the mass of human transferrin produced in insect (Drosophila melanogaster S2) cells is consistent with lack of sialic acids on its N-glycans (Lim et al., 2004). 2. N-Linked Glycans a. Analysis of derived glycopeptides and site occupancy. Although N-linked glycans have long been known to attach to a consensus sequence consisting of an Asn-Xxx-Ser(Thr) motif, where Xxx can be any amino acid except proline, evidence is now emerging that Asn-Xxx-Cys can also act as a motif. Satomi, Shimonishi, and Takao (2004) have identified a third site based on this motif, occupied by approximately 2% of the glycosylation, in human transferrin.

Proteolysis, usually with trypsin, to localize each consensus sequence site to an individual glycopeptide is the classical method for determination of the glycosylation at each site. Detection of glycopeptides by HPLC following proteolysis is often difficult because of effects such as signal suppression. Monitoring of oxonium ions (163 for hexose, 204 for GlcNAc, etc.) is a standard method for tracking the glycopeptides and has been extended to monitoring with an ion trap mass spectrometer by generating them in the ion-source region (Sullivan, Addona, & Carr, 2004) . Peptide masses were confirmed by MALDI-TOF MS after desalting with C18 ZipTips. A method for predicting peptide retention times in reversed-phase HPLC has been published (Krokhin et al., 2004b) following examination of 346 tryptic peptides and deglycosylated glycopeptides identified by MALDI-TOF MS from 17 proteins. Glycopeptides eluted slightly earlier than their deglycosylated versions. Liu, Feasley, and Regnier (2004) have evaluated the technique of diagonal chromatography which involves comparisons of HPLC chromatograms run under identical conditions before and after enzymatic removal of the glycans and have concluded that, although the technique worked well for detection of protein phosphorylation, retention times of the glycopeptides and derived peptides, identified by MALDI-TOF MS, were too similar for diagonal chromatography to be a reliable technique for detecting glycopeptides. MALDI-TOF MS has the advantage over ESI-MS for the detection of glycopeptides in that the spectra usually only contain singly charged ions unlike ESI with its tendency to produce multiply charged ions. Glycopeptides are often revealed in MALDI profiles by their relatively high mass and by peak spacing corresponding to monosaccharide residues (e.g., 162 for hexose) (Krokhin et al., 2004a) .

Rather than attempt detection of glycopeptides in the presence of peptides, some investigators employ fractionation techniques to isolate the glycopeptides before mass spectrometric analysis. For example, Wada et al. (2004) have developed a method whereby glycoproteins are reduced and alkylated, cleaved with trypsin and lysylendopeptidase and partitioned with cellulose or Sepharose to bind the glycopeptides. The isolated glycopeptides could then be examined by LC or linear MALDI-TOF. MS/MS in an ion-trap-TOF instrument provided peptide fragments for protein identification and carbohydrate ladders that gave information on the glycan composition. The method was applied to transferrin, IgG and b 2 -glycoprotein 1. A method has been reported for identification and quantification of N-linked glycoproteins following their specific immobilization on a solid support . Cis-diol groups in the carbohydrate were oxidized with periodate to aldehydes allowing the carbohydrate to be captured by hydrazide chemistry. The protein was then cleaved with trypsin or another suitable enzyme and the resulting peptides were released with PNGase F for analysis by MALDI or ESI mass spectrometry. Deuterium labeling of the peptide with succinic anhydride allowed quantitative studies to be made.

The frequent absence of glycopeptides in LC/MS analyses has been used to advantage in indicating which peptide might be glycosylated. Thus, peptides containing the consensus N-glycosylation sites at Asn-142, 173, and 178 were not observed by Hambrock et al. (2004) from the tryptic digest of the recombinant protein TSC-36/Flik expressed in human cells suggesting that these sites were glycosylated. An et al. (2003b) have used pronase to digest two wellcharacterized glycoproteins and found that steric factors prevented complete digestion in the region of the glycosylation site leaving the carbohydrate attached to a short peptide. N-glycan masses were obtained by MALDI-FT MS from a separate experiment involving glycan removal by PNGase F and subtracted from those of the glycopeptides to obtain the peptide mass and, hence, its structure. The method was claimed to be more rapid that the traditional method of proteolysis and the results easier to interpret because no large peptides appeared in the final sample. The method was applied to the study of site heterogeneity of Xenopus laevis egg cortical granule lectin.

Asn to Asp conversion after PNGase digestion effectively labels the percentage of a given site that is occupied and is detected by a mass difference of one unit. The technique has been used, for example, to show glycosylation in 10 of the 11 consensus sequence sites of the SU component of avian sarcoma/ leukosis virus envelope glycoprotein (Kvaratskhelia et al., 2004) and to identify occupied glycosylation sites in membrane-bound glycoproteins. Normally this latter task is difficult because 2D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels are ineffective in separating these compounds. To overcome the problem, the proteins were solubilized in guanidine-HCl, digested with trypsin and the glycopeptides were recovered by lectin binding. Following removal of the N-glycans with PNGase F, the peptides with the Asn to Asp conversion were detected by MALDI-Q-TOF MS (Fan et al., 2004) . Investigations of the spike glycoprotein from the SARS

virus have revealed four occupied and one unoccupied N-linked sites by molecular weight measurements made before and after glycan removal by PNGase F and by observing the Asn to Asp conversion (Ying et al., 2004) . In a similar experiment, digestion of the glycoprotein N-CAM with AspN and trypsin resulted in the identification of glycopeptides, each containing a single glycosylation site. They were separated by HPLC and analyzed by MALDI-TOF MS. Five of the six sites were identified but glycopeptides from site Asn-4 were missing. This site was eventually characterized by a direct MALDI-FTICR analysis of the glycopeptides released in-gel by trypsin (Albach et al., 2004) . Incorporation of 18 O into the aspartic acid from H 2 18 O can be used to label the newly produced aspartic acid and was used by Barinka et al. (2004) to identify and confirm glycosylation on each of the 10 consensus sequence sites of glutamate carboxypeptidase II.

b. N-Linked glycan composition from glycopeptide analysis. Measurement of glycan masses directly or by subtraction of the mass of the peptide from those of glycopeptides leads directly to the constituent isobaric monosaccharide composition as illustrated by a recent study of N-glycan masses from the androgenic hormone of Porcellio scaber (Crustacea) (Grève et al., 2004) . Sometimes, with large glycopeptides, peaks are poorly resolved but can often be improved by chemical treatments. Thus, for example, the core fragment of human luteinizing hormone gave a broad, poorly resolved peak (m/z 8,700-10,700) when examined from sinapinic acid with a linear TOF instrument (Jacoby, Kicman, & Iles, 2003) . The compound consisted of two peptide chains, only one of which was glycosylated, linked with four disulfide bonds. These bonds were reduced on the MALDI target with dithiothreitol (DTT) to give a more fully resolved envelope of peaks from which the N-linked glycan composition was deduced by subtraction of the peptide masses.

c. Glycan release. Most structural determinations of these compounds are performed on released glycans. Experimental details for chemical and enzymatic release of N-glycans have been described (Merry & Astrautsova, 2003) . Release with PNGase F (or PNGase A if the glycans contain fucose in a-(1 ! 3)-linkage to the core GlcNAc) or hydrazine are the preferred enzymatic and chemical release methods respectively. No new chemical release techniques or modifications were found during the review period reflecting the increasing popularity of enzymatic release, particularly with PNGase F. Additionally, PNGase F release is not accompanied with the potential hazards of hydrazine. However, there is some controversy as to whether this enzyme releases all glycans, particularly from larger glycoproteins. Thus, it has been shown not to release Glc 1 Man 9 GlcNAc 2 (28) from arylophorin under non-denaturing conditions and, in addition, Reinhold et al. (Zhu et al., 2004a) assert that PNGase F fails to release a subset of glycans from C. elegans and thus use hydrazine. Artifacts detected by MALDI-TOF analysis after in-gel release with PNGase F have included glycans with urea attached to the reducing terminus (Omtvedt et al., 2004) as the result of its inclusion in an earlier procedure for glycoprotein purification. Deglycosylation with trifluoromethanesulfonic acid, a method that leaves the protein intact for further study has been reviewed (Edge, 2003 using the well-studied glycoproteins a1-antitrypsin and ribonuclease B. Gutternigg et al. (2004) , however, preferred ATT as the matrix because of its neutrality. A large number of studies have been conducted on the structural determination of N-glycans from isolated glycoproteins and from intact organisms or tissues; these are summarized in Tables 2 and 3 respectively. Among the more unusual glycosylations to have been reported are the presence of Glc 1 Man 9 GlcNAc 2 (28) in the insect Antheraea pernyi (Chinese oak silkworm) Complex, di-, tri-, tetra-antennary Peptides, not glycosylation control residence time β 

Site-specific glycan characterization. (Kamar, et al., 2004) α1-Acid glycoprotein Human PNGase F R-TOF (DHB), Glycans and Me esters Complex Altered glycosylation in patients with inflammation and diabetes. (Higai, et al., 2003a) α1-Acid glycoprotein Human, ox, sheep, rat PNGase F Trypsin, chymotrypsin L-TOF (DHB), Glycans

Comparison of glycosylation in four species. Different glycosylation in each. (Nakano, et al., 2004) α1-Acid glycoprotein Human PNGase F Trypsin L-+ R-TOF (DHB), Glycans (various hydrazones)

High-mannose hybrid, complex Use of cationic or hydrophobic derivs. to enhance signal α1-Acid glycoprotein Human PNGase F TOF, Glycans (2-AA)

Complex, fucosylated di-and tri-antennary New separation method. Fucglycans up in cancer (Szöllosi, et al., 2004) α1-Acid glycoprotein 

Cancer patients showed raised AGP with abnormal glycans Aminopeptidase N (Stephens, et al., 2004b) α1-Antitrypsin Identification of congenital disorders of glycosylation α1-Antitrypsin Human hepatoma cells

Complex Increase in 1→3-fucosylation in hepatoma cell line (Higai, et al., 2003b) α1-Antitrypsin -PNGase F Trypsin TOF (DHB), Glycans

Complex (di-, tri-, tetra-antennary) As reference compound to demonstrate on-target digestion Amyloid fibril Human Trypsin TOF, Glycopeptide Complex Detection of one occupied glycosylation site (Karimi, Sletten & Westermark, 2003) Androgenic hormone

Structural identification (Grève, et al., 2004) α1-Antitrypsin Under-glycosylation of plasma α1-antitrypsin not random (Mills, et al., 2003a) TABLE 2. Use of MALDI MS for examination of N-glycans from specific glycoproteins (see also Asialofetuin Fetal calf serum PNGase F Trypsin L-+ R-TOF (DHB), Glycans (hydrazones) Complex Use of cationic or hydrophobic derivatives. to enhance signal Avidin Complex Glycan identification in human ovarian tumour marker N-Cadherin High-mannose, complex Structural characterization CD24 (Heat stable antigen)

PNGase F R-TOF (DHB), Glycans Complex Structural determination from different cell lines (Ohl, et al., 2003) Chorionic gonadotropin Complex Abnormal (branched on 3-arm) biantennary glycans in choriocarcinoma JEG-3 cells Cortical granule lectin

PNGase F Pronase, Trypsin

High-mannose, complex Asn-154, 217 N-Glycan ident. method based on pronase digestion to determine site occupancy Cytotoxin 3 Naja kaouthia (Cobra) venom PNGase F Trypsin L-, R-TOF (DHB, Sinapinic acid) Glycopep.

Complex, Asn-29 First ident. of glycosylation in three-fingered toxin (Osipov, et al., 2004) Decay-accelerating

Structural identification (Lukacik, et al., 2004) Ecto-5'-nucleotidase Bull seminal plasma CNBr, trypsin L-, R-TOF (CHCA),

Oligo-Man, hybrid, complex, Asn-403

Structural identification (Fini, et al., 2003) Site-specific structure determination. (Hyuga, et al., 2004) α-Galactosidase (Caputo, et al., 2003) Glutamate carboxypeptidase

Trypsin, Asp-A TOF (CHCA, ferulic acid, DHB), Glycopeptides

All 10 sites occupied but nature of glycans not stated Identification of glycosylation sites by 18 O-incorporation (Barinka, et al., 2004) β 2 -Glycoprotein I Human Trypsin, Lysylendopeptidase TOF (DHB), MS n Complex As test compound for method based on hydrophilic affinity isolation (Wada, et al., 2004) Gp17/SABP Human seminal fluid PNGase F Trypsin SELDI, L-TOF (CHCA) CID Glycans (Per-Me)

Different N-linked glycan profile in pathological state (Caputo, et al., 2003) GP120 HIV-I sf2 T r y p s i n L-TOF (CHCA, +ve, DHB, -ve), LC/MS (ESI), Glycopeptides Complex, high-Man, 110, 154, 160, 170, 203, 214, 235, 249, 274, 304, 328, 334, 358, 370, 378, 415, 428 High-mannose complex (Gal-Fuc) Structural identification. Novel galactose-substitution on core fucose (Wuhrer, et al., 2004c) Hemocyanin Megathura crenulata (Keyhole limpet) Hydrazine MALDI, Glycans (2-AP)

High-mannose, hybrid First Gal-β-(1→6)-Man and Fuc-α-(1→3)-GalNAc epitopes Hemocyanin High-mannose, complex Structural identification mainly by HPLC. IgD/IgE Human PNGase F, Ingel R-TOF (DHB), HPLC (2-AB)

Identification of glycans. Interaction with lectins. (Arnold, et al., 2004) IgG Human (Sjögren's disease)

Hydrazine TOF, Glycans, (ABEE) Complex Structural ident. Lower Gal on glycans from patient (Kuroda, et al., 2002) IgG Human Hydrazine R-TOF (DHB), MALDI-Q-TOF, LC/MS (2-AB)

Hybrid, complex

Identification of congenital disorders of glycosylation IgG α-Gal-caps) Structural identification IgG Human Trypsin, Lysylendopeptidase TOF (DHB), MS n Complex As reference compound for method based on hydrophilic affinity isolation (Wada, et al., 2004) IgM Determination of site-specific glycosylation 

Endo-D, PNGase F TOF (sinapinic acid) Complex Fuc-GlcNAc on Asn-54 needed for max. IL-10R2 binding (Logsdon, et al., 2004) Integrin

PNGase F TOF, (+ve) (DHB),

High-mannose, complex Glycans from melanoma integrins have β1→6-branches (Pochec, et al., 2003) (Westphal, et al., 2003) Lysozyme mutant (G49N)

PNGase F (In gel)

High-mannose plus GlcNAc Yeast shown to be capable of adding GlcNAc to glycans (Yoko-o, et al., 2003) Matrix metalloproteinase-9

Human PNGase F, trypsin TOF, Glycans Complex (biantennary) Structural determination. Asn-28, 120 (Kotra, et al., 2002) Neoculin Curculigo latifolia fruit -

Paucimannosidicdeduced from mass Structural characterization of taste-modifying protein (Shirasuka, et al., 2004) Neural cell adhesion molecule Calves Endo N PNGase F Trypsin TOF (ATT), ESI, Ion trap, Glycans (2-AP)

Complex HNK-1 Epitope -sites 2,4,5,6

Structural identification. Polysialic acid removed by Endo N treatment (Wuhrer, et al., 2003a) Ovalbumin Chicken PNGase F L-+ R-TOF (DHB), ESI, Procyclin Trypanosoma brucei HF, PNGase F TOF (CHCA), Glycopeptides, Glycans

High-mannose Identification of glycosylation mutants resistant to Con A Prostate Hybrid, complex Structure of glycans in normal and prostate cancer (Ohyama, et al., 2004) Receptor (Gotte, et al., 2003) Ribonuclease B Bovine pancreas PNGase F Trypsin L-+ R-TOF (DHB), Glycans (hydrazones)

High-mannose Use of cationic or hydrophobic derivs. to enhance signal Ribonuclease B Bovine pancreas 

To investigate the effect of charged (sialylated) glycans on glycoprotein migration in isoelectric focusing gels Thy-1 Avian in Lec1 or Tn5 cells Asp-N, Glu-C TOF (CHCA), glycopeptides Paucimannosidic Structural identification (Mehndiratta, et al., 2004) Transferrin Human PNGase F R-TOF (DHB, 3-AQ), Glycans (Me ester, ANTS)

Multiple changes in glycosylation explains poor sensitivity as EtOH-use marker (Flahaut, et al., 2003) Transferrin Human serum Identification of congenital disorders of glycosylation Transferrin Structural identification. Glycosylation at Asn-491 in Asn-Xxx-Cys motif. (Satomi, et al., 2004) Transferrin Human Trypsin, Lysylendopeptidase TOF (DHB), MS n Complex As reference compound for method based on hydrophilic affinity isolation (Wada, et al., 2004) Transferrin Human hepatoma cells PNGase F R-TOF (DHB), Glycans Complex Increase in (1→3)-fucosylation in hepatoma cell line (Higai, et al., 2003b) Vicilin Cor a 11

Trypsin TOF, Glycopeptides

To study allergenic behaviour. (Lauer, et al., 2004) Vitelline

PNGase F

High-mannose, complex Study of the effect of glycan structure on sperm-binding (Vo, et al., 2003) Vitellogenin High-mannose Structural identification (Khalaila, et al., 2004) β-Xylosidase High-Man (Me-Man) paucimannosidic Structural identification (Gutternigg, et al., 2004) Caenorhabditis elegans (Natsuka, et al., 2002) Caenorhabditis elegans Soluble glycoproteins Hydrazine TOF (DHB), Glycans (2-AP)

High-mannose, truncated complex Structural identification and genetics (Hirabayashi, et al., 2002) Chinese hamster

Ovary cells with GlcNAcT-III PNGase F L TOF (s-DHB), Glycans

Complex (with bisect) Structural identification. GlcNAc-TIII produces bisects (Lee, Park & Stanley, 2003b) Chinese hamster, (wild and Lec19

Ovary cells

PNGase F TOF (+ve, -ve) (s-DHB, THAP), Glycans

High-mannose, complex Lec19 CHO cells have reduced ability to add galactose (Lee, et al., 2003c) Chinese hamster, (Schmitt, et al., 2004a) Human Serum Hydrazine R-TOF (DHB), Q-TOF, LC/MS, Glycans (2-AB)

High-mannose, hybrid, complex

Identification of congenital disorders of glycosylation Maize Glycoproteins PNGase A R-TOF (DHB), Glycans (Reduced, Per-Me) Paucimannosidic For detection of immunogenic core (1→3)-Fuc and core Xyl (Bardor, et al., 2003a) Mice Erythrocytes Hydrazine TOF (DHB)

Complex β-Gal-transferase-1 deficient mice produced more N-Ac-lactosamine (Kotani, et al., 2004) Nicotiana benthamiana Paucimannosidic, highmannose, complex Structural identification. Glycans similar to those of higher plants (Viëtor, et al., 2003) Oryza sativa (Rice)

PNGase A R-TOF (DHB), Glycans (Reduced, Per-Me) Paucimannosidic For detection of immunogenic core (1→3)-Fuc and core Xyl (Bardor, et al., 2003a) Rice, maize, wheat, date

PNGase A L-TOF, R-TOF (DHB/HIQ), GC/MS, Glycans Paucimannosidic N-Glycan structure similar between mono-and di-cotyledons (Léonard, et al., 2004) . Paucimannosidic glycans with similar Gal-b-(1 ! 4)-Fuc and Gal-b-(1 ! 4)-Gal-b-(1 ! 4)-Fuc groups at the reducing-terminal 6-position have been found in keyhole limpet (Megathura crenulata) hemocyanin (Wuhrer et al., 2004c ). An unusual difucosylated core structure Man 3-GlcNAc 2 dHex 2 ) in which one of the fucose residues is attached to a mannose residue has been reported as a constituent of phaiodactylipin (phospholipase A 2 ) from scorpion Anuroctonus phaiodactylus) venom (Valdez-Cruz, Batista, & Possani, 2004) . High-mannose glycans including the first report of a glycan with a Gal-b-(1 ! 6)-Man moiety have been reported from keyhole limpet hemocyanin together with glycans with a Fuc-a-(1 ! 3)-GalNAc epitope which causes cross-reactivity with Schistosoma mansoni glycans which share same epitope . Some N-glycans from C. elegans and related organisms are substituted with phosphorylcholine (PC, 3/13) which is thought to modulate the host immune response. Studies on their biosynthesis have shown that C. elegans microsomes transfer PC from L-a-dipalmitoylphosphatidylcholine (31) to both hybrid and complex N-glycans containing GlcNAc (Cipollo et al., 2004a) . HL60 cells treated with the glucosidase inhibitor n-butyldeoxynojirimycin (NB-DNJ, 32) produced a series of compounds with the structure of high-mannose glycans but with only one GlcNAc residue as the result of cytosolic cleavage from misfolded glycoproteins that had been exported from the endoplasmic reticulum (Mellor et al., 2004b) . A novel trisulfated hybrid glycan containing a GlcA residue capping the Gal-GlcNAc antenna has been identified from bovine myelin glycoprotein P0 (Yan, Kitamura, & Nomura, 2003) .

e. Applications of MALDI MS to the study of N-glycan function. The presence of N-glycans on ribonuclease has been shown to reduce oligomerization (Gotte, Libonati, & Laurents, 2003 

O-Linked glycans continue to receive less attention than their N-linked counterparts, one factor being their tendency to occur in groups that make site analysis difficult. As with N-linked glycans, much structural analysis is conducted on released glycans. A method for the identification of sites modified by O-GlcNAc that relies on mild b-elimination followed by Michael addition with dithiothreitol and known as BEMAD has been described (Wells et al., 2002) . The method was validated by mapping three previously identified O-GlcNAc sites, as well as three novel sites, on Synapsin I purified from rat brain and on the Lamin B receptor and the nucleoporin Nup155.

a. Studies on intact glycoproteins and glycopeptides. Collision-induced decomposition (CID) spectra of glycopeptides obtained by ESI from doubly charged ions frequently display prominent loss of the sugar moieties as the result of the labile protons. Czeszak et al. (2004) have used MALDI-PSD on glycopeptides derivatized with a phosphonium group at the amino-terminus and have shown that the resulting singly charged phosphonium ions and a-type fragments retain the sugar. In contrast, when doubly charged ions were generated by proton addition, loss of the glycan was again seen. The method was claimed to be an effective alternative to electron-capture dissociation (ECD) and was used to locate up to three GalNAc residues on the full tandem repeat peptide derived from the MUC5AC mucin. Kurogochi, Matsushita, and Nishimura (2004) have reported that LIFT-TOF with DHB as the matrix is an ideal system for fragmenting glycopeptides (b and y series ions) and have used it to determine the O-linked glycosylation sites in mucin-type glycopeptides. Up to six O-linked glycosylation sites have been found in the hinge region of IgA1 from human serum . Tryptic digestion yielded a 33-mer glycopeptide that was examined by MALDI-TOF MS from THAP/ammonium citrate. This matrix gave more highly resolved spectra than s-DHB or ANALYSIS OF CARBOHYDRATES AND GLYCOCONJUGATES & CHCA; the CHCA spectrum, in particular, was very poorly resolved . Structural analysis was by exoglycosidase digestion (sialidase and galactosidase) and site analysis was performed on the degraded glycans by further cleavage with the endoproteases Glu-C from Staphyloccus aureus and proteinase K from Tritirachium album . MALDI-TOF analysis of the stalk-region glycopeptide (APTPVPPPTGTPRPL) from murine CD8 has shown that all three threonine residues are glycosylated with HexNAc. Four peaks were obtained and attributed to molecules containing 0-3 additional hexose residues .

The 47 kDa portion of the 45/47 kDa glycoprotein from Mycobacterium tuberculosis expressed in Streptomyces lividans has been found to contain glycans on both the N-and C-terminal tryptic peptide; MALDI-TOF analysis showed from 0 to 9 hexose residues at the N-terminus but the glycosylation site was not determined (Lara et al., 2004) . The 13-amino acid-containing peptide isolated from Conus textile contains a Gal-GalNAc residue attached to threonine but with an unknown linkage between the sugar rings. MALDI-TOF analysis of the products of a b-galactosidase digest on a synthetic analogue containing the common b-D-Gal-(1 ! 3)-a-D-GalNAc-carbohydrate showed that the enzyme was unable to remove the galactose residue suggesting the presence of a1 ! 3-linked galactose and this was confirmed by subsequent NMR analysis (Kang et al., 2004) . Tryptic glycopeptides from surface glycoproteins of the hepatitis B virus were found by MALDI-TOF MS and on-target exoglycosidase digestion to be O-glycosylated with Gal-GalNAc or Neu5Ac-Gal-GalNAc. The N-glycans were mainly biantennary complex (Schmitt et al., 2004a) .

b. Applications of MALDI to the structural determination of O-linked glycans. Studies on the structural determination of O-glycans from isolated glycoproteins and from intact organisms or tissues are summarized in Tables 4 and 5 respectively. Studies on some specific structural types are summarized below.

i. Mucins. The ability of MALDI MS to resolve complex mixtures of glycans has been utilized in studies of mucins from the jelly coats of amphibian eggs. Thus, three layers of the jelly coat from X. laevis have been shown to contain different but overlapping glycan structures; 40 neutral and 30 sulfated compounds were identified by HPLC (PGC column), MALDI-FT-ICR, CID and the ''catalog-library'' approach of CID peak matching reported earlier (Tseng, Hedrick, & Lebrilla, 1999) . Thirty-five neutral, sulfated and sialylated glycans have been identified from X. tropicalis by similar techniques combined with exoglycosidase digestion (Zhang et al., 2004a) . Sialylated glycans were stabilized by formation of methyl esters. Sulfated core-2 and core-4 glycans have been identified by MALDI-TOF MS and NMR from respiratory mucins from a patient suffering from chronic bronchitis but, unlike the structures of glycans identified earlier from a cystic fibrosis patient (Lo-Guidice et al., 1994) , these glycans contained no sialic acid (Degroote et al., 2003) . MALDI-TOF data were recorded in negative ion mode and no loss of sulfate was reported. Human intestinal mucins show an acidic gradient along the intestinal tract which may explain the region-specific colonization of the gut by various bacteria (Robbe et al., 2003b) . O-Glycans of stomach mucosa have an antibacterial action against Helicobacter pylori by inhibiting biosynthesis of cholesteryl-a-D-glucoside (identified by MALDI-TOF MS), a major cell wall component (Kawakubo et al., 2004) .

ii. Glycosaminoglycans (GAGs). Methods for the analysis of hyaluronan and its fragments have been reviewed (Capila & Sasisekharan, 2004) .

a. Unsulfated GAGs. Hyaluronan oligomers (

, with masses up to 8 kDa, obtained from hyaluronic acid by the action of bovine testicular hyaluronidase have been examined by electrospray and MALDI-TOF MS. Electrospray showed the presence of oligomers with both odd and even numbers of sugar units whereas MALDI and high-performance anion exchange chromatography (HPAEC) showed only even numbered oligomers. The discrepancy was traced to the electrospray ion source which was producing cone-voltage fragmentation. It was recommended that for ESI studies of compounds of this type, the cone voltage be kept low and precisely controlled (Prebyl et al., 2003 ). An LC/ MS method for quantification of hyaluronic acid fragments in pharmaceutical preparations produced by the action of hyaluronate lyase from Streptococcus agalactiae has been reported, with negative ion MALDI-TOF from DHB being used to record the glycan profiles of three fractions. The largest fraction showed peaks to 15 kDa (Kühn et al., 2003) . A model disaccharide, DUA-GlcNAc (10 mmol), derived from heparan sulfate by heparitinase enzyme digestion and bearing an unsaturated 4,5uronic acid (DUA) at the non-reducing end has been adducted to mercuric acetate and the product characterized by MALDI-TOF MS, confirming the formation of a mercury adduct (m/z calc. 639.9, obs. 640 with the expected spread of 6 mass units for Hg 198 to Hg 204 , consistent with the production of a cyclic 4,5mercurinium intermediate (Skidmore et al., 2004) .

b. Sulfated GAGs. A common method for analysis of these glycans is the use of basic peptides for ion pairing as outlined in the earlier reviews in this series. Thus, for example, cleavage of heparin by controlled g-irradiation has produced fragments enriched in highly sulfated sequences which were examined by MALDI-TOF MS using ion-pairing with (Arg-Gly) 19 -Arg to stabilize the sulfates (Bisio et al., 2004) . Studies by MALDI-TOF MS and HPLC of the serine protease inhibitor and its chondroitinase and hyaluronidase digestion products have shown that, in acute inflammation, its chondroitin-4 sulfate chain is both longer and undersulfated compared with control glycoproteins (Capon et al., 2003) . Combined MALDI-TOF and enzyme digestion have also been used by Kett and Coombe (2004) for GAG analysis.

A method for simultaneous analysis of both N-and O-linked glycans has been published by Robbe et al. (2003a) . Glycans were released by non-reductive b-elimination using the ammonia-based method described earlier by Huang, Mechref, and Novotny (2001) (Plihal, et al., 2004) Bile Core-1 core-2 Structural identification.

Branched core I in cancer C1 Inhibitor

Human in rabbit mammary gland β-Elimination R-TOF (DHB (+ve) THAP/NH 4 citrate), Glycans

Core-1 Structural ident. of glycans expressed in milk (Koles, et al., 2004b) Carbohydrate-

Trypsin, chymotrypsin TOF (Cinnamic acid), SELDI, Glycopeptides Mannose Structural identification. 3 Regions of glycans with maximum of four mannose (Boraston, et al., 2003) κ-Casein Neu5Gc-Gal-GalNAc Structural identification (Mamone, et al., 2003) κ-Casein

Trypsin, GluC

Neu5Gc-Gal-GalNAc Structural identification (Holland, Deeth & Alewood, 2004) CD8 Mouse Trypsin R-TOF (DHB), ESI, Glycopeptides

Core-1

Structural identification Chorionic gonadotrophin Human in CHO cells Hydrazine R-TOF (DHB CMBT), SSI, Ion trap, Glycans (2-AB) 2 to 6 residues, Sialylated Structural identification (Gervais, et al., 2003) Coagulation Core-1 Structural identification (Lukacik, et al., 2004) Granulocyte- Ser-5,9, Thr-10, core 1

Structural identification (Forno, et al., 2004) IgA ( Core II Structural identification, mainly by HPLC 

2-aminoacridone (AMAC, 1/58) or 8-aminonaphthalene-1,3,6trisulphonic acid (ANTS, 34). They were then separated by gel electrophoresis, the bands containing the glycans were excised and the derivatized glycans were extracted for analysis by MALDI and electrospray MS. Comparisons between the electrophoresis profiles from the AMAC (uncharged) and ANTS (charged) derivatives provided an indication of the charge state of the glycan. The method was designed mainly for profiling mucin glycans and was applied to porcine gastric mucin and bovine submaxillary mucin.

A popular method for the independent study of both N-and O-glycosylation is to remove the N-linked glycans with, for example, PNGase F and then examine the O-glycosylation, either by further release using b-elimination or by measurements on the glycopeptides obtained from the O-linked region (Forno et al., 2004; Trimble et al., 2004) . The well-established differential hydrazinolysis method was used by Gervais et al. (2003) to study N-and O-linked glycosylation of human recombinant chorionic gonadotropin allowing O-linked glycans to be identified for first time.

With the recent sequencing of several genomes, attention has become focused on how the complexity of higher organisms can be encoded by such a small number of genes. Post-translational modifications and, in particular, glycosylation have emerged as important determinants of the control of biological systems. Lamarre-Vincent and Hsieh-Wilson (2003) have studied glycosylation of cyclic AMP-responsive element-binding protein, a transcription factor essential for long-term memory and have shown the first link between O-GlcNAc and information storage in the brain. O-GlcNAc was detected by specific enzymatic radiolabeling with galactose from [ 3 H]UDP-galactose to the 4position of O-GlcNAc. MALDI-TOF analysis of the tryptic peptides was used to show that the enzyme was modified with two O-GlcNAc residues.

Most organisms synthesize a-linked-polyglucans, such as glycogen, as an energy source when other reduced carbon compounds are insufficiently available but, to date, the method for the initiation of glycogen synthesis in prokaryotes has not been determined. In an attempt to rectify this gap in our knowledge, Albrecht et al. (2004) have produced the eukaryotic enzyme yeast glycogenin (Glg2p), a known initiator in fungi and animals, in E. coli and found it to be autocatalytically glucosylated at tyrosine residues 232 and 230 with from 4 to 25 glucose residues, as determined by MALDI-TOF and MS/MS analysis. Further incubation with UDPglucose resulted in transfer of more than 36 additional glucose residues over 20 min as detected by MALDI-TOF. In another study, the 

z Under-O-glycosylation in IgA nephropathy (Horie, et al., 2003a) IgA1 Human serum Identification of glycans.

Computer program to interpret data IgA1 Selenoprotein P

Glu-C Trypsin L-TOF (CHCA), Glycopeptides

Thr-346, Hex, HexNAc, Neu5Ac

Structural identification (Ma, et al., 2003a) Zona pellucida 3

Human and mouse β-Elimination R-TOF (DHB), Glycans (Per-Me) Gal, GalNAc, Neu5Ac, Neu5Gc Proteins from both sources have same glycans (Coppin, et al., 2003) Caenorhabditis elegans Total glycoproteins β-Elimination TOF, Q-TOF (DHB), Glycans

GlcA, Gal, GalNAc Srf-3 Mutant, resistant to bacterial infection is deficient in glycoconjugates (Cipollo, et al., 2004b) Hepatitis B virus

Trypsin, O-glycosidase TOF (ATT), Glycopeptides GalNAc-Gal-Neu5Ac Structural identification (Schmitt, et al., 2004a) Human Airway mucin

Core-2, core-4 Biosynthesis of airway mucins related to bronchial disease (Degroote, et al., 2003) Human Intestinal mucins β-Elimination TOF (DHB), GC/MS, Glycans

Core-5 Evidence for region-specific glycosylation dependent on pH (Robbe, et al., 2003b) Human Tonsillar lymphocytes Trypsin TOF, glycopeptides GalNAc, Gal, Neu5Ac Asialo-O-glycans deposited in IgA nephropathy (Hiki, et al., 2004) Mouse Intestinal mucins β-Elimination TOF, GC/MS, Glycans (N-Meamide)

Core-1, core-2 Alterations in intestinal mucins caused by parasite (Holmén, et al., 2002) Xenopus laevis

Egg mucin β-Elimination FT-ICR (DHB (+ve) 2,5-DHA (-)), CID, Glycans

Neutral, sulfated, core-2 Structural determination. Catalog library approach Ox Submaxillary gland mucin Neutral, acidic, unspecified core

Non-reductive β-elimination with NH 4 OH allows labeling (Robbe, et al., 2003a) Rat Skeletal muscle (Zhang, et al., 2004a) N-terminal segment of potato X virus has been found to be glycosylated at the N-terminus with galactose or fucose and that the presence of the sugar mediates the formation of a bound water shell on the virion surface (Baratova et al., 2004) .

Glycan profiling by MALDI-TOF MS for the detection of disease is gaining ground. For example, a new method has been developed for the extraction of the acute-phase glycoprotein, a1-acid glycoprotein from human serum and its N-glycans have been compared in patients with inflammation or cancer and healthy controls. The disease states produced an increase in both the degree of branching and in the amount of fucosylation Szöllosi et al., 2004) . The amounts of fucosylated bi-, tri-, and tetra-antennary glycans were also found to increase at the expense of unfucosylated triantennary glycans in this glycoprotein in patients with acute inflammation (Higai et al., 2003a) . Sialylated glycans were stabilized for MALDI-TOF analysis by formation of methyl esters. The same stabilization method was used by Flahaut et al. (2003) in a study on the effect of ethanol on transferrin glycosylation in chronic alcoholics has revealed patient-dependent differences in both the level of sialylation and in the number of glycans attached to the molecule, possibly accounting for the controversial use of transferrin glycosylation as a marker for chronic ethanol consumption.

a. Detection of cancer biomarkers. N-glycans from normal human pancreatic cells consist mainly of complex biantennary structures with small amounts of tri-and tetra-antennary compounds and larger compounds with poly-N-acetyllactosamine extensions and extensive fucosylation (up to five fucose residues and eight Gal-GlcNAc units as detected by MALDI-TOF MS). Glycans from ribonuclease extracted from pancreatic adenocarcinoma tumor cells, on the other hand, contain fucosylated hybrid structures and biantennary glycans whose antennae terminated in GalNAc, thus providing a potential diagnostic test for the adenocarcinoma (Peracaula et al., 2003a) . MALDI-TOF analysis gave much better resolution of the large glycans than could be obtained by HPLC. Altered glycosylation has also been found associated with prostate cancer; glycans from prostate-specific antigen (PSA) contained increased amounts of fucose and GalNAc in the antennae, again indicating a possible diagnostic test (Peracaula et al., 2003b) . Another study found increased amounts of a-(2 ! 3)-linked sialic acid in human polysialic acid (PSA) suggesting that differential binding to Maackia amurensis agglutinin lectin could be used to differentiate prostate cancer from benign prostate hypertrophy (Ohyama et al., 2004) . Masses of some sialylated glycans were reported from linear MALDI-TOF spectra but most measurements were made on neutral glycans following linkage-specific removal of sialic acid. Increased fucosylation has also been found in N-linked glycans from human hepatoma (Higai et al., 2003b) and melanoma (Ciolczyk-Wierzbicka et al., 2004) cell lines and a reduction in the concentration of fucosylated glycoproteins has been found in dogs undergoing treatment for lymphosarcoma (Xiong, Andrews, & Regnier, 2003a ). An extensive study of the N-and O-glycosylation of the human ovarian tumor marker CA125 has been reported. The bi-, tri-, and tetra-antennary glycans were identified by MALDI-TOF, MS/MS and FAB mass spectrometry as permethylated derivatives coupled with exoglycosidase digestion . Highly processed glycans have also been found on the breast tumor marker protein, gross cystic disease fluid protein-15, than on the same protein from healthy controls (Caputo et al., 2003) . Glycans were again examined as permethylated derivatives but, this time, by surface-enhanced laser desorption/ionization (SELDI) mass spectrometry. MUC1 glycoprotein is overexpressed in breast cancer and O-Glycans have been shown to control proteolysis by preventing proteolysis of the Thr3-Ser4 bond if either amino acid is glycosylated (Hanisch et al., 2003) . Tetrasialylated-tetra-antennary glycans carrying a b1 ! 6 antenna appear to be associated with the development of cancer metastases as demonstrated by studies on integrins from metastatic and non-metastatic human melanoma cell lines (Pochec et al., 2003) . MALDI-TOF profiles were obtained following in-gel glycan release using the method described by Küster et al. (1997) as modified by Hoja-Lukowicz et al. (2000) . Bi-and tri-antennary glycans have been identified attached to human chorionic gonadotropin produced in a choriocarcinoma cell line (JEG-3); the biantennary glycans, which were useful markers, were unusual in having a branched 3-antenna (35) rather than carrying both unbranched 3-and 6-antenna . The structure was confirmed by MALDI-TOF analysis of exoglycosidase digestion products with an a-mannosidase digestion following b-galactosidase digestion to confirm the presence of the exposed mannose residue on the 6-antenna. The study illustrates the danger of assigning structures directly by matching masses with those from a database or by using only the usual exoglycosidase sequence of b-galactosidase followed by N-acetylhexosaminidase which would incorrectly have suggested a normal biantennary structure such as 23.

b. Congenital diseases of glycosylation. Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) analysis of both the N-glycans released from tryptic peptides and of the peptides themselves has shown that the asparagine residues of a1-antitrypsin in patients suffering from congenital disorder of glycosylation (CDG) type I (deficiency in the initial protein glycosylation by Glc 3 Man 9 GlcNAc 2 , 36) are preferentially glycosylated in the order Asn-46 > 247 > 83 (Mills et al., 2003a) . Asparagine occupation was measured by observing the extent to which it was converted to aspartic acid during glycan removal by the enzyme PNGase F. In a newly characterized disorder, CDG-1i, MALDI-TOF and HPLC were used to show that some of the N-glycans on fibroblasts consisted only of Man 1 GlcNAc 2 and Man 2 GlcNAc 2 (Thiel et al., 2003) . Analysis by MALDI-TOF and HPLC of serum glycoproteins from several patients with CDG type II syndromes (defective glycan processing) has been used to identify several enzyme defects. One patient was characterized with a mannosidase-III deficiency that prevented biosynthesis of complex glycans as the result of failure to remove residual mannose residues from the 6-antenna. The predominant hybrid glycan 37 was characterized by CID using a MALDI-Q-TOF instrument . a-Mannosidase deficiency causes accumulation of mannosecontaining oligosaccharides (released from N-glycans) in various tissues but the disease has been successfully treated by enzyme replacement therapy. Residual glycans were monitored in this study by MALDI-TOF MS (Roces et al., 2004) . A GlcNActransferase-II deficiency in another patient in the study by Butler et al. prevented c. Other diseases. The hinge region of IgA1 has been shown by MALDI-TOF MS to be under-O-glycosylated in patients with IgA neuropathy (Horie et al., 2003a) . As upper respiratory tract infection often precedes hematurea and proteinurea in these patients, Horie et al. (2003b) have investigated the effect of tonsillectomy and steroid treatment and found that they produce increases in the extent of IgA O-glycosylation. Again, analysis was by MALDI-TOF with s-DHB as the matrix. A method has been reported for detection of the biomarker, serum amyloid component, in human plasma or urine. Extraction was by monoclonal antibody binding and analysis by MALDI-TOF MS from sinapinic acid. Several new, desialylated compounds were detected from three individuals (Kiernan et al., 2004) . Krokhin et al. (2003) have identified, in a preliminary study, several high-mannose and complex N-glycans from the spike protein of the SARS virus taken directly from patients. The compounds were identified mainly with the aid of MS/MS on a Q-TOF instrument.

Work with these compounds usually involves analysis of intact proteins that are modified with chemically attached glycan residues and are ideal subjects for the high mass resolving ability of MALDI-TOF instruments. Analysis of b-lactoglobulin glycation products formed from lactose and galactose has revealed lysine as the main binding site with additional binding at the amino-terminal leucine residue and at Arg-124. MALDI-TOF analysis was found to be more efficient than LC/ESI-MS for locating the binding sites (Fenaille et al., 2004) . MALDI-TOF has, for the first time, enabled kinetic data to be obtained on defined glycation products from specific sites of a glycated protein. Thus, lysozyme was incubated with D-glucose for 1, 4, 8, or 16 weeks in phosphate-buffered saline, digested with endoproteinase Glu-C and the C-and N-terminal peptides were used for the relative quantification of the initial Amadori product, N e -(carboxymethyl)lysine (38) and an imidazolone (39) formed from arginine (Kislinger et al., 2003) . The preferred glycation sites in human serum albumin (HSA) have been determined as lysines 233, 276, 378, 525, and 545 following incubation of HSA with glucose, enzymatic digestion of the protein with either trypsin or endoproteinase Lys-C, and examination of the resulting glycated peptides by LC/MS/MS and MALDI-TOF from CHCA (Lapolla et al., 2004a) . Glycation of casein by glucose, fructose or ribose has been characterized and shown not to affect its protective effects on human intestinal Caco-2 cells (Jing & Kitts, 2004) . A study of 20 Type 2 diabetic patients and 10 controls have shown that complications of diabetes are associated with a higher concentration of glyco-oxidized variants of globin than observed in controls or patients without complications (Lapolla et al., 2004b) .

Several other studies on advanced glycation end products (AGEs) have been reported. Yamada et al. (2004) have synthesized a collagen model peptide and subjected it to glycation with glucose, ribose and glyoxal (40). A dimeric peptide linked glyoxal-lysine dimer (41) was isolated and identified by MALDI-TOF as the first example of an intact glycation-derived dimer. The same technique has been used to show that methylglyoxyl, produced during the Maillard reaction binds to Arg-184 and Arg-185 located within the binding site of bovine glutathione peroxidase and to deactivate the enzyme, resulting in an increase in the intracellular peroxidases responsible for oxidative cellular damage . AGEs from ribose were formed more rapidly when incubated with bovine serum albumin (BSA) than those from glucose or fructose as measured by binding to AGE receptors (Valencia et al., 2004) . The use of in-house-developed PERL script software has been used to identify and localize AGEs on b 2 -microglobulin . The program identifies masses, measured by MALDI-TOF MS that correspond to those of peptides with an AGE modification. In other studies, the AGEs N e -(carboxymethyl)-lysine, imidazolone A (42) and B (43), pyrraline (44) and 1-alkyl-2-formyl-3,4glycosyl pyrrole (45) have been found on the type 2 ryanodine receptor calcium-release channel, accounting for decreased cardiac contractility in diabetes . The soluble AGE receptor (sRAGE), a member of the immunoglobulin superfamily, has been characterized from mouse and its Nglycosylation identified by MALDI-TOF MS (Hanford et al., 2004) . In another study, measurements of protein molecular weights have added support to the conclusion that Amadori decomposition pathways are constrained in the presence of metal ion chelators and radical traps (Culbertson et al., 2003) .

Although protein glycation is usually associated with pathology, as in the secondary effects of diabetes, the reaction is also involved in cooking where it changes the color (browning) and flavor of the food. Fay and Brevard (2004) have recently reviewed the use of mass spectrometry in the study of the involved Maillard reaction.

One protein encoded by the 13 Drosophila peptidoglycan recognition proteins has been shown to possess enzymatic activity. MALDI-TOF analysis of the degradation products demonstrated that the enzyme hydrolyses the lactylamide bond between the glycan chain and the cross-linking peptides. The enzyme was, thus, classified as an N-acetylmuramoyl-L-alanine amidase (Mellroth, Karlsson, & Steiner, 2003) . HPLC and MALDI-PSD were used by Antignac et al. (2003) to characterize the peptidoglycan from Neisseria meningitidis. The peptidoglycan was hydrolyzed with muramidase from Streptomyces globisporus. Fourteen of the 28 muropeptides separated by HPLC were found to be O-acetylated to different degrees. Proteoglycan-like glyconectins from three sponges, Microciona prolifera, Halichondria panicea, and Cliona celata have been examined by NMR and mass spectrometry. The structures were complicated and contained distinct acid-resistant and acid-sensitive domains. Some sugars were sulfated and others pyruvylated (Guerardel et al., 2004) . Muropeptides derived from Escherichia coli peptidoglycan have been labeled with the fluorescent dyes ANTS, 7-aminonaphthalene-1,3-disulfonic acid (ANDA, 46) or 4-aminonaphthalene-1-sulfonic acid (ANSA, 47) and examined by fluorophore-assisted carbohydrate electrophoresis (FACE). Results compared favorably, both qualitatively and quantitatively with earlier HPLC-based methods. MALDI-TOF MS was used to confirm the identity of the FACE separated glycans (Li, Höltje, & Young, 2004d 

Some small anaerobic bacteria such as treponemes contain glycoconjugates that are different from the normal glycolipids. One of these glycoconjugates has been isolated from the oral spirochete, Treponema medium by phenol/water extraction and the carbohydrate removed enzymatically for structural identification by NMR and MALDI-TOF MS. Fragmentation was performed with a TOF/TOF instrument and revealed the glycan sequence of a tetrasaccharide repeat unit containing the two amino acids ornithine (48) and Asp. The repeat unit (

that was amide-linked to L-ornithine and fucose linked to Daspartic acid (Hashimoto et al., 2003a) . 

Many examples of the application of MALDI-based methods to analysis of these compounds have been published; these are summarized in Table 6 and only a few representative studies and some with more unusual features will be discussed here. Extractions are normally carried out with hot aqueous phenol. These compounds often contain many more monosaccharide constituents than are found in the glycoproteins, requiring additional techniques such as combined gas chromatography/ mass spectrometry (GC/MS) and NMR for their analysis. Their generally acidic nature, conferred by Kdo glycans and phosphate groups, makes them ideal candidates for examination in negative ion mode.

a. Intact LOS. Although small lipooligosaccharides can be examined directly, most have to be modified by hydrolysis of the large O-glycan chain or by removal of much of the lipid component of the lipid A (1/18) moiety. Thus, for example, the backbone structures of the LOS from V. parahaemolyticus strains have been obtained following O-and N-deacylation with hydrazine and dephosphorylation with TFA (Hashii et al., 2003a,b) . Laser-induced decomposition can be a problem or, in some cases, can yield useful structural information. Thus, the negative ion MALDI-TOF spectrum from DHB of the shortchain LOS from a strain of V. parahaemolyticus (3 kDa) following deacylation showed successive losses of phosphoethanolamine (49), hexuronic acid and phosphate (Hashii et al., 2003b) . LOS from M. catarrhalis has been deacylated with hydrazine and examined in negative linear mode from DHB/ hydroxy-iso-quinoline (HIQ). The spectrum showed several peaks produced by in-source cleavage at the labile Kdo-lipid A glycosidic bond (Luke et al., 2003) . The lipid A molecule itself, examined in reflectron mode, was found to contain seven acyl groups and varying amounts of ethanolamine esterified to the two phosphate groups. Both MALDI-TOF and nanospray MS/MS analysis of the de-O-acylated (hydrazine) LOS from Pseudoalteromonas haloplanktis TAC 125 grown at 158C has shown the presence of three phosphate groups, two on the lipid A and one on the core oligosaccharide, whereas at 258C, an additional phosphate was added to the core (Ummarino et al., 2003) . The LOS from Pseudoalteromonas issachenkonii KMM 3549 T has been found to contain what is thought to be the first example of a 4,7-disubstituted heptose as determined mainly by NMR; MALDI-TOF MS was used to delineate the lipid A acylation pattern (Silipo et al., 2004b) . Two sets of ions were obtained as the result of cleavage of the labile Kdo-lipid A bond. 

Haemophilus ducreyi has been shown to be capable of incorporating sialic acids with N-acyl groups larger than acetyl. However, the extent of sialylation dropped with increasing chain length, becoming non-existent for sialic acid with chains above C8 (Goon et al., 2003) . The LOS was deacylated with hydrazine and examined by MALDI-TOF from DHB/HIQ in negative ion mode; positive ion MS/MS spectra were obtained with electrospray ionization.

b. Lipid A. Mild acid hydrolysis of the labile bond between Kdo and the lipid A moiety is the normal method for releasing lipid A from these glycolipids. A method combining thin-layer chromatography (TLC) with both MALDI-TOF and ESI-MS n (up to MS 4 ) has been applied to the analysis of lipid A from E. coli. The predominant fragmentation was loss of acyl groups but, whereas this appeared to occur by charge-remote processes in the CID spectra, charge-driven processes appeared to predominate in the MALDI spectra (Lee et al., 2004a) .

Several novel lipid As have been discovered during the review period. The first example of a neutral lipid A has been isolated from the predatory bacterium Bdellovibrio bacteriovorus (Schwudke et al., 2003) . The molecule consisted of a b-(1 ! 6)linked 2,3-diamino-2,3-dideoxy-D-glucopyranose disaccharide carrying six hydroxylated fatty acids. a-D-mannopyranose residues occupied the two positions normally occupied by phosphates. A similar 2,3-diamino-2,3-dideoxy-D-glucopyranose (Preston, et al., 2003) Burkholderia caryophylli of Ara4N at reducing end of lipid A (Molinaro, et al., 2003) Burkholderia cepacia EPS L-TOF (+ve, -ve) (DHB), NMR -α-D-Gal, β-D-Gal, Kdo Structure. Growth in mannitol-enriched medium gave →6)-β-DFruf-(2→ (levan) (Cescutti, et al., 2003) Butyrivibrio fibrisolvens H10b (Andersson, et al., 2003) Chlamydophila psittaci 6BC LPS Lipid A L-TOF (THAP + 0.1% TFA), NMR 5 α-GlcN, β-GlcN, Kdo Structure. LPS less active than typical endotoxins in cytokine induction. Chlamydia trachomatis E, L 2 LPS Lipid A L-TOF (THAP + 0.1% TFA), NMR 5 α-GlcN, β-GlcN, Kdo Structure. LPS less active than typical endotoxins in cytokine induction. Coxiella burnetii GlcpN Lipid As with different degrees of Ac more potent than fully acylated (Mueller, et al., 2004) Francisella novicida in E.

coli

L-TOF (ATT) 6 GlcN, Kdo Lipid A 1-de-phosphorylation occurs on periplasmic surface of inner membrane (Wang, et al., 2004c) Haemophilus ducreyi LPS TOF (DHB/HIQ) -Neu5Ac homologues,

β-D-GlcNAc H. ducreyi can incorporate unnatural sialic acids into LPS (Goon, et al., 2003) (Tran, et al., 2004) Klebsiella pneumoniae essential for LPS synthesis (Regué, et al., 2004) Leptospira interrogans 

Lipid Structural characterization (Corsaro, et al., 2004) Pseudomonas stutzeri OX1 Core TOF (-ve) (THAP), NMR -GlcN, GalN, Hep, Kdo, Glc, pyruvic acid Structural determination of novel highly negatively charged LPS (Leone, et al., 2004a) Pseudomonas stutzeri OX1 (Hashii, et al., 2003b) Xanthomonas campestris pv.

pruni Lipid A L-, R-TOF (THAP) 6 GlcNAcp, phosphate Presence of methyl-branched acyl chains (Silipo, et al., 2004d) Yersinia pseudotuberculosis (Marceau, et al., 2004) disaccharide carrying six hydroxylated fatty acids was found in the lipid A from Leptospira interrogans which causes a hemorrhagic fever known as Weil's disease. The molecule carried only one phosphate group which was mono-methylated. The two hydroxy-acyl groups attached to the diamino-glucose residue I were esterified with unsaturated o-6-12:1 and 14:1 fatty acids (Que-Gewirth et al., 2004). 2,3-Diamino-2,3-dideoxy-Dglucopyranose has also been found in lipid A from Bartonella henselae, a Gram-negative bacterium that causes cat-scratch disease (Zähringer et al., 2004) and in that from Mesorhizobium huakuii. In the latter compound, the phosphate group normally attached to the di-NH 2 -Glc moiety II was replaced by D-glucuronic acid (Choma & Sowinski, 2004) . A 4-amino-4deoxy-L-arabinopyranose-1-phosphate has been found at the reducing end of the lipid A from Burkholderia caryophylli (Molinaro et al., 2003) . The negative ion MALDI-TOF (DHB) spectrum showed considerable heterogeneity due to differing numbers of acyl groups. The proximal (reducing end) GlcN residue of lipid A from Rhizobium leguminosarum lacks a phosphate group and can be oxidized to 2-amino-2-deoxy gluconate by an enzyme recently identified in the outer membrane of the bacterium (Que-Gewirth et al., 2003). The first example of lipid A from an Agrobacterium species (A. tumefaciens strain C58, a plant pathogen) has revealed the presence of a normal bis-phosphorylated glucosamine disaccharide backbone with several acyl variants. The main species was a penta-acyl derivative bearing two 3-OH-14:0 fatty acids in ester linkage and two 3-OH-16:0 acids in amide linkage. The 3-OH-16:0 acid on GlcN II was esterified with 27-OH-28:0 that, in turn was esterified with a 3-OH-butyroyl residue . Other lipid A molecules to have been analyzed in the review period are listed in Table 6 .

c. Core oligosaccharide. Mild acid hydrolysis is also the method normally used to obtain core oligosaccharides from LPS. Ethanolamine diphosphate has been identified for the first time and its position of substitution established in the LPS core of a rough-type mutant of Pseudomonas aeruginosa (PAO1 DalgC). In addition, the paper reported a reinvestigation of the structure of the complete LPS core of wild-type P. aeruginosa PAO1 and a reassignment of the phosphorylation sites (Kooistra et al., 2003) . The LPS was complex because of the presence of esterified fatty acids that were removed by mild hydrazinolysis prior to analysis by ESI and MALDI. Phosphoethanolamine has also been found in strains of N. meningitidis and 4,6-O-(1-carboxy)-ethylidine residues derived from pyruvic acid have been identified in the core of LOS from Pseudomonas stutzeri OX1 (Leone et al., 2004a) .

The O-chain from Bordetella avium LPS, obtained by mild acid hydrolysis to remove the lipid A followed by hydrofluorolysis to remove the core oligosaccharide, was found to consist only of a chain of the substituted 1 ! 4 linked monosaccharide 2-acetamido-3-(3-hydroxybutanamido)-2,3dideoxy-b-D-glucopyranosyluronic acid (50). The negative ion MALDI-TOF spectrum from DHB showed a series of peaks from 1 to 3.8 kDa (Larocque et al., 2003) . Other examples of O-chain analysis are cited in Table 6 . Table 6 and only two representative examples will be expanded here. The structure of the repeating unit of the EPS from Butyrivibrio fibrisolvens strain H10b is a hexasaccharide carrying O-acetyl and 1-carboxyethyl groups and has been determined with a variety of techniques including NMR and circular dichroism (CD) following extraction with phenol. MALDI-TOF MS of fractions from a partial acid hydrolysate was used to determine the carbohydrate sequence (Andersson, Cotta, & Kenne, 2003) . B. cepacia, a bacterium causing lung infections was obtained from a cystic fibrosis patient and cultured on agar plates. Phenol was added to the harvested cells, stirred for 5 hr at 48C and the EPS was precipitated with isopropanol. MALDI-TOF and NMR were used to determine the structure of the repeating unit as

. When grown on a mannitol-rich medium, conditions that induce the formation of EPS, the bacteria produced a second compound with the structure ( ! 6)-b-D-Fruf-(2 ! ) (Cescutti et al., 2003) .

a. Studies on the glycan moiety following removal of the ceramide. Glycosphingolipids can be analyzed directly by MALDI-TOF but the spectra are usually complicated because of heterogeneity in the ceramide portion of the molecule. Thus, many investigators use an enzyme such as ceramide glycanase to remove the ceramide. A recent example is a study of the effect of N-alkyl analogues of deoxynojirimycin (DNJ), inhibitors of ceramide glucosyltransferase, on GSL structure. GSLs were cleaved with ceramide glycanase and labeled with 2-AB (1/56) for analysis by HPLC and MALDI-TOF (Mellor et al., 2004a) . Carbohydrates substituted with phosphocholine (PC) and phosphoethanolamine (PE) have been released with endoglycoceramidase from glycosphingolipids of the pig parasitic nematode Ascaris suum and identified by MALDI, ESI, GC/ MS (methylation analysis) and NMR (Friedl et al., 2003) .

A rapid method for splitting glycosphingolipids into their three components (sugar, sphingosine and fatty acid) by use of a domestic microwave oven has been reported by Itonori et al. (2004b) . The glycosphingolipids were heated with 0.1 M NaOH/ MeOH for 2 min followed by 1 M HCl/MeOH for 45 sec. The alkaline methanolysis step produced the intermediate lysoglycosphingolipids virtually free of by-products such as the Omethyl ethers that are usually seen but the N-acetylamino-sugars needed re-acetylation by reaction with acetic anhydride/pyridine at room temperature.

b. Studies by TLC and MALDI. Glycosphingolipids are frequently separated by TLC. Ivleva et al. (2004) have published a method for attaching TLC plates to a MALDI target and have obtained spectra of even fragile compounds such as gangliosides by using an FTICR instrument fitted with a vibrationally cooled MALDI ion source. Twenty matrices were investigated and the soft matrices, DHB, ATT and anthranilic acid (1/57) were found to produce the least sialic acid loss from gangliosides. TLC resolution was best preserved by using matrices in rapidly evaporating organic solvents.

c. Applications to the structural elucidation of glycosphingolipids. A novel GlcNAc-a-1 ! HPO 3 ! 6-Gal-(1 ! 1)-ceramide (51) has been identified from the liver fluke, Fasciola hepatica, by MALDI-TOF and electrospray-MS/MS. HF treatment released the GlcNAc residue, confirming the GlcNAcphosphate link (Wuhrer et al., 2003b) . Mammalian type globoand iso-globotriaosylceramides have also been found in this species obtained from infected sheep. MALDI-TOF spectra were obtained from both the intact glycosphingolipids and the 2-AP derivatives of their released glycans using ATT as the matrix, and the presence of antigenic terminal a-galactose was demonstrated by on-target incubation with a-galactosidase (from chicken) (Wuhrer et al., 2004a) . Ceramides carrying b-Gal-(1 ! 6)-b-Gal-(1 ! 1)-Cer and larger oligosaccharides with from one to three additional b1 ! 6-linked mannose residues represent a departure from the common glycosphingolipids that contain inositol phosphate found in many fungi and may account for the resistance of this species to the fungicide, Aureobadidin A . Measurements by SELDI MS of tetanus neurotoxin (53.7 kDa) incubated with the ganglioside GT1b, and observation of the intact protein/ganglioside complex, has shown that two carbohydrate binding sites are required for toxicity (Rummel et al., 2003) .

d. Miscellaneous studies. Two photoaffinity probes (ganglioside GM2 labeled on the acyl chain with a p-(3-trifluoromethyl)diazirinyl-phenyl group) (52), were used to probe the active site of GM2-activator protein. After binding, and trypsinization, the tryptic peptides were examined by MALDI-TOF and ESI-MS and found to be bound to the most flexible region of the protein (Wendeler et al., 2004a) . g-Radiolysis of glucosylceramide and galactosyl diglyceride resulted in the release of the sugars as shown by MALDI-TOF MS (Shadyro et al., 2004) . Other studies and details of the above work are summarized in Table 7 . 

Novel zwitterionic glycosphingolipids having the sugar chain capped with phospholcholine have been reported from the mycelia of filamentous fungi of Acremonium species . Structures were of the type PC ! 6-(Man-(a1 ! 6)-Man-(a1 ! 6)-GlcN-(a1 ! 2)-Ins1-P-1-Cer. A related compound without the attached glycan chain has been identified from the red alga Gracilaria verrucosa (Khotimchenko & Vas'kovsky, 2004) .

The glycoglycerolipid a-D-Glcp-(1 ! 3)-a-D-Glcp-(1 ! 1)-lipid where the lipid is glycerol containing a C 15 acyl group at C-2 and a C 15 ether at C3 has been separated from the propionibacterium Propionibacterium propionicum by TLC and its structure determined by MALDI-TOF, NMR, ESI/MS, and GC/MS. Related compounds acylated at C6 of the outer glucose residue and compounds lacking either this glucose or one fatty acid residue were also found (Pasciak et al., 2003) . Glycoglycerolipids from the thermoplilic bacterium Meiothermus taiwanensis have been shown to have the structure a-Galp-(1 ! 6)-b-Galp-(1 ! 6)-b-GalNAcyl-(1 ! 2)-a-Glc-(1 ! 1)Gro where Acyl ¼ 17:0 or hydroxy-17:0 (Yang et al., 2004) . A similar compound from the thermophilic bacterium Thermus oshimai NTU-063 has the structure b-Glcp-(1 ! 6)-b-Glcp-(1 ! 6)-b-GlcpNAcyl-(1 ! 2)-a-Glcp-(1 ! 1)-glycerol diester where the N-acyl group is a 15:0 or 17:0 fatty acid and the glycerol-attached fatty acids have chain lengths from 15:0 to 18:0. The MALDI-TOF spectrum contained a range of peaks from m/z 1,300 to 1,550 (Lu et al., 2004a) . Di-mannosyl-acyl monoglyceride in which the mannose residue adjacent to the glycerol contained an anteisobranched 15-carbon acid at the 6-position has been characterized from Rothia mucilaginosa, a pathogen responsible for several opportunistic infections. Unusually the acyl group attached to the glycerol was at C2 rather than the more usual C3 (Pasciak et al., 2004) .

The structure of a novel mannose-capped lipoarabinomannan from Amycolatopsis sulphurea has been investigated with a range of techniques of which MALDI-TOF (DHB, negative ion) showed an average molecular mass of around 10 kDa but with a broad, unresolved envelope of peaks with a half-height width of approximately 5 kDa . The lipoarabinomannan from the mycobacterium-like organism Tsukamurella paurometabola has produced a MALDI-TOF spectrum from s-DHB with a broad peak with a mass range from 9 to 16 kDa. The structure was mainly determined by NMR. It lacked some of the features such as branching arabinan chains that are found in arabinomannans from mycobacteria but, nevertheless the molecule was claimed to be the most elaborated non-mycobacterial arabinomannan identified to date . Lipomannan and lipoarabinomannan from Mycobacterium kansasii has been shown to possess a manno-oligosaccharide cap but to lack the phosphoinositol cap found in lipoarabinomannans from Mycobacterium smegmatis (Guérardel et (Batrakov, et al., 2004) Ascaris suum Not analysed Structural identification of zwitterionic glycans containing phosphocholine and phosphoethanolamine (Friedl, et al., 2003) Cow and human (Bode, et al., 2004) Fasciola hepatica (liver fluke) Mammalian-type glycolipids.

Gal by exoglycosidase digestion (Wuhrer, et al., 2004a) Human Vitreous body of eye TOF (DHB), TLC (Fujiwaki, et al., 2004) Human and rat Classification into new molluseries (Aoki, et al., 2002) Mucor hiemalis Plasmodium falciparum Intra-erythrocyte stage L-, R-TOF (DHB, norharmane), TLC Gal, Glc, GalNAc d18:0, d20:0 OH-10:0, OH-12:0, OH-14:0 Structural determination (Couto, et al., 2004) (Korduláková et al., 2003; .

Mass spectrometric methods for analysis of flavonoids have been reviewed (Prasain, Wang, & Barnes, 2004) . Three glycosyltransferases (Pérez et al., 2004b) and two methyltransferases (Pérez et al., 2004a) involved in the biosynthesis of phenolic glycolipids from Mycobacterium tuberculosis have been characterized. MALDI-TOF spectra of the glycolipids showed a range of produces with masses of around 1.5 kDa resulting from acyl heterogeneity. A new mannitol teichoic acid has been reported from the cell wall of the bacterium Brevibacterium sp. VKM Ac-2118 isolated from a frozen late Pliocene layer (1.8-3 Myr) of Kolyma lowland, Russia. The structure consisted of 1,6-poly(mannitol phosphate) with the majority of mannose residues carrying additional phosphate groups. Positive ion MALDI spectra from DHB gave peaks to 6 kDa (Potekhina et al., 2003) . The structures of hexamannosides from mycobacterial species have been elucidated and found to contain up to four long-chain acyl groups (Gilleron, Quesniaux, & Puzo, 2003) . A typical structure is 53. Studies with M. smegmatis have shown that mannose metabolism, as revealed by studies on phosphoinositol mannosdes, is required for growth (Patterson et al., 2003) . Mycoglycolipids have been isolated from the edible fungus Hypsizygus marmoreu and identified by GC/MS and MALDI-TOF MS . The biosynthesis of mycobacterial phosphatidyl mannosides has been revised with the help of MALDI-TOF analysis (Morita et al., 2004) . PIM 4 (four mannose residues and two palmitate chains) present in mycobacterial phosphatidyl manno-oligosaccharide, identified by PSD from CHCA, has been shown to be a natural antigen for CD1d-restricted T cells .

Although MALDI-TOF MS is used quite extensively in this field, FAB still appears to be the most commonly encountered ionization technique. Glycosides identified by MALDI during the review period are listed in Table 8 . Glucosylated heteropolyflavans (e.g., 54) from Sorghum bicolor have been examined by Krueger, Vestling, and Reed (2003) ; polymers with from three to seven flavan units and up to seven glucose residues were found. To overcome problems with estimation of the number of hydroxyl groups present due to the coincidence in mass with the [M þ K] þ ion, samples were analyzed as cesium adducts by addition of cesium trifluoroacetate to the sample/matrix solution. Silver trifluoroacetate was also investigated but problems were Ardisia mamillata

Triterpenoid saponins TOF, FAB, ORD, NMR, LC β-D-Glcp, α-L-Rhap, α-LArap Borrelia burgdorferi

Cholesteryl galactoside L-TOF, NMR, FAB, GLC β-D-Gal, Agent for Lyme disease (Ben-Menachem, et al., 2003) Borrelia burgdorferi (Schröder, et al., 2003) Cheilanthes glauca (Neretina, et al., 2002) Henricia sanguinolenta and H. leviuscula leviuscula Steroidal polyols. Cholesterol TOF (CHCA), HPLC, TLC, NMR Mono-, di-and tri-OMe-β-Xylp (sanguinoside C from starfish) (Levina, et al., 2003) Henricia sanguinolenta and H. leviuscula leviuscula

Steroidal glycosides (Cholestanes, cholestenes) TOF (CHCA), NMR, HPLC, TLC 2-O-Me-Xylp Hippasteria phrygiana (Starfish)

Steroidal glycosides (Cholestanes, cholestenes) TOF (CHCA), NMR, HPLC, TLC β-D-Xylp, α-L-Araf Hordeum sp. (Barley) Flavone glycosides TOF (DHB), NMR β-D-Glc (Nørbaek, et al., 2003) Lupinus oreophilus Ten triterpenoid saponins FT-ICR, ESI, NMR, HPLC α-L-Ara, β-D-Gal, β-D-Glc, α-L-Rha (Woldemichael, Montenegro & Timmermann, 2003) Marrubium velutinum Acetylated glycosides FT-ICR (DHB) LC TLC NMR β-D-Glcp (Karioti, et al., 2003) Mycale laxissima Steroidal glycosides TOF, NMR, GC/MS D-Ara, D-Glc, D-Gal (Antonov, et al., 2003) Mycobacterium tuberculosis,

Phosphatidyl-myo-inositol mannoside R-TOF (-ve) (HABA), NMR β-D-Man Phlomis physocalyx Hub.-Mor.

Phenylethanoid tetraglycoside (physocalycoside) FT-ICR (DHB), MNR, OR, IR, UV, TLC α-L-Rha, β-D-Glc, Api (Ersöz, et al., 2003) Plantago major L.

Phenolic glycoside, (Verbascoside,

TOF, NMR, HPLC Glc, Rha (Egorov, et al., 2004) 

Triterpene glycoside TOF (CHCA), NMR Xyl, Qui, OMe-Glc Quillaja saponaria

Triterpenoid saponins (28 compounds)

β-Gal, β-GlcA, α-L-Rha, β-Fuc, α-L-Ara, β-Xyl, β-Glc, β-D-Api (Nyberg, et al., 2003) Rhizophora mangle L. Flavonol glycosides from tannin MALDI Rha, Glc, (Mangrove) (Kandil, et al., 2004) Sorghum bicolour (L.) Moench

Heteropolyflavans L+R-TOF (+ve) (3-IAA), Glc (Krueger, et al., 2003) Solenostemma argel (Asclepiadaceae)

Oxo-pregnane glycosides (stemmosides C and D) R-TOF (CHCA), NMR, ESI Linear pentasaccharide, β-D-Glcp β-D-Olep, β-D-Canp, β-D-Cymp (Plaza, et al., 2004) 

Triterpenoid saponins TOF (CHCA), NMR, GC/MS β-D-OMe-Glcp, β-Xyl, β-D-Quip, β-4-SO 3 Na-Xyl (Moraes, et al., 2004) Thalictrum orientale

Phenyl glycosides FT-ICR, UV, NMR β-D-Glcp, β-D-Xylp (Erdemgil, et al., 2003) (Nyberg, Baumann, & Kenne, 2003) . The triterpene glycoside from the sea cucumber Stichopus mollis (Stichopodidae) was found by Moraes et al. (2004) to differ in structure from the corresponding glycosides from all other known stichopodids in possessing a sulfate group in the carbohydrate chain, prompting the authors to propose a reclassification to a new genus, Australostichopus Levin. Both MALDI and ESI in negative ion mode have been demonstrated to be capable of detecting carminic acid (55), the red dye isolated from cochineal, in paintings applied to canvas. Binding agents such as linseed oil did not interfere with the detection (Maier, Parera, & Seldes, 2004) . MALDI-TOF has also been used to study the biotransformation of ginsenoside Rb 1 by 49 microbial strains (Dong et al., 2003) . 

These compounds are mixtures of cyclic, branched, and linear polymers of glucose containing various species-different substituents. They accumulate in the periplasmic space of gramnegative bacteria in response to low osmolarity. Lequette et al. (2004) have identified the gene that encodes for the protein that controls the backbone structure of these carbohydrates. Associated analysis of the carbohydrates with masses up to 3.5 kDa was performed by MALDI-TOF from DHB or 3-AQ.

Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has been used by many laboratories to monitor the products of enzyme reactions aimed at deducing mechanisms of action or characterization of newly discovered enzymes. Much of this work is summarized in Table 9 . Desorption/ionization on silicon mass spectrometry (DIOS-MS) or laser desorption without a matrix has been shown to be a viable alternative to MALDI for analysis of small molecules and carbohydrates and has been used to monitor the activity of a2 ! 6-sialyltransferase on the disaccharide Gal-b-(1 ! 4)-GlcNAc (Shen et al., 2004) . Jobron et al. (2003) have immobilized N-acetyllactosamine on a cellulose membrane and used it as an acceptor for assaying four glycosyltransferases. The trisaccharide produced by a1 ! 3-galactosyltransferase was identified by MALDI and the method was proposed as the basis of a high-throughput technique for enzyme screening. A method for detecting the presence of O-glycanase, an enzyme that cleaves the Galb-(1 ! 3)-GalNAca-(1 ! ) link to serine or threonine, uses MALDI to detect the [M þ Na] þ ion at m/z 406 corresponding to the released sugar (Akai et al., 2003) .

A MALDI-TOF method for the simultaneous detection and measurement of sugars, ascorbic acid (57), citric acid (58), and sodium benzoate has been developed by Ayorinde, Bezabeh, and Delves (2003) . The matrix was meso-tetrakis-(pentafluorophenyl)porphyrin (59), a compound with a molecular weight of 974 which is well above that of the target molecules. Sodium or potassium hydroxides were used as dopants. The method was particularly good at detecting the low molecular weight organic acids. MALDI-TOF MS, from DHB also provides a method for detecting the presence of starch hydrolysates as adulterants in fruit juices (Cabálková et al., 2004) . The action of the enzyme preparation, Olivex, used in the preparation of olive (Olea europaea) oil has been investigated. The preparation is mainly a pectinase that hydrolyses (1 ! 4)-linked galactan chains from the cell wall. However, it is unclear how this reaction improves the quality of the product (Vierhuis et al., 2003) . A comparative study of five Rhizobium strains from different cropping areas of China has concluded that differences in the efficiency of nitrogen fixation is not related to differences in Nod factor structure (identified by MALDI-TOF and ESI-Q-TOF MS) (Thomas-Oates et al., 2003) . 

Production of humanized antibodies and other biopharmaceuticals in plants and insect cell lines, as a safe and economically feasible alternative to production in animals, requires reengineering of the N-glycan synthesis machinery to eliminate potentially antigenic a1 ! 3-fucose-and b1 ! 2-xylose-containing glycans. MALDI-TOF analysis is ideally placed for analysis of the resulting glycans and many examples are listed in Table 10 . An analysis of human transferrin expressed in Lymantria dispar (gypsy moth), however, has shown the natural absence of core a1 ! 3-fucosylation, a normally common motif in insects (Choi et al., 2003b) . To study the specificity of IgG and IgE antibodies against the a1 ! 3-fucose and b1 ! 2-xylose epitope, Bencúrová et al. (2004) have modified the relatively simple biantennary glycans on human transferrin to incorporate these epitopes. Products were monitored by MALDI-TOF MS and antibody binding was reported to be greatest for the fucosylated glycans. Maeda et al. (2004) have replaced all of the asparagine residues of the N-glycosylation sites in procarboxypeptidase Y by alanine and expressed the protein in P. pastoris. The resulting protein lacked N-glycosylation but contained residual glucose, thought to be O-linked, as detected by an increase in the MALDI mass from that expected from the amino acid sequence. Analysis of erythropoietins (EPO) with varying glycosylation has shown the fully sialylated tetraantennary glycan, identified by HPLC and MALDI-TOF MS as its 4-AA derivative, to be the major determinant of biological activity (Yuen et al., 2003) . Sanz-Nebot et al. (2003) have recorded MALDI spectra from intact human recombinant EPO and noted a drop in measured mass with increasing laser power which they attributed to loss of sialic acid within the ion source. MALDI-TOF has been used to identify O-glycans from a von Willebrand factor C domain produced in P. pastoris (O'Leary et al., 2004) . Methods for their removal were investigated.

A method for monitoring glycosylation in pharmaceuticals based on capillary electrophoreses of 3-AA derivatives with structural confirmation by MALDI-TOF MS has been published by Kamoda et al. (2004) . MALDI and ion exchange chromatography have proved useful for checking the batch-to-batch variation in glycosylation of three recombinant gonadotropins. As an example, excellent consistency (3.5) was found for the degree of sialylation of 120 batches of recombinant thyroid stimulating hormone over a 3-year period (Gervais et al., 2003) .

Several reviews on the production of biopharmaceuticals and containing reference to MALDI-TOF analysis have been published. These include production of human therapeutic proteins in yeasts (Bretthauer, 2003) , yeasts and filamentous fungi (Gerngross, 2004) and lepidopteran cell lines (Tomiya et al., 2004) . Methods for characterizing biological products after manufacturing changes have also been reviewed (Chirino & Mire-Sluis, 2004) .

Slime deposits in paper mills, termed biofouling, is a problem in the paper industry and has prompted studies on the composition of their glycolipids. The EPS from Methylobacterium strains, responsible for ''pink slime'' have been shown by Verhoef et al. (2003) to consist of the repeating unit:

Sadly, many chemical papers appear to ignore details of the equipment and conditions used to obtain mass spectra of carbohydrates (and other compounds) even though conditions such as the choice of MALDI matrix are often essential in getting the compounds to ''fly.'' Although NMR experiments and equipment are usually described in great detail, information on mass spectrometry is frequently reduced to uninformative acronyms such as HRMALDIMS with no further details. Without knowledge of the equipment used to record the spectra it is difficult to judge if HR is indeed meaningful. The relegation of essential methods to ''supplementary information'' is also to be regretted. The absence of essential information such as the matrix from many publications is reflected in Table 11 (with apologies to authors from whose papers this information has been missed). ''MALDI'' is used for papers omitting to cite the type of instrument used to record the spectra. Most of the publications in this area relate to routine monitoring of reaction products and are summarized in Table 11 . In addition to chemical reactions, enzymes are frequently used in this area because of their stereospecificity. Both transferases and glycosidases such as PNGase F used in their reversed mode to catalyze transglycosylations can be used; however, evidence has been found to suggest that enzymes used for reverse hydrolysis reactions can have their activity reduced by Maillard glycation (Maitin & Rastall, 2004) . As with the previous review, TOF, HPLC (Kaneko, et al., 2003) Human, GnT-IX Transfers GlcNAc to the 6-antenna of N-glycans. Also transfers GlcNAc to the 6-position of the 3-antenna to produce a novel structure. Enzymes uniquely expressed in brain.

R-TOF (DHB), NMR (Inamori, et al., 2003) Human, GnT-IX Enzyme acts on the GlcNAc-β-(1→2)-Man-α-1-Ser/Thr moiety forming a 2,6-branched structure in brain O-mannosyl glycan.

R-TOF (DHB), NMR (Inamori, et al., 2004) pp α-GlcNAc-T2 (Korekane, et al., 2003) Saccharomyces cerevisia A strain of this yeast is unusually shown to be able to add a single GlcNAc residue to a variety of high-mannose glycans. In-gel removal of glycans with PNGase F. TOF (DHB) (Yoko-o, et al., 2003) 

GalNAc-transferase 2 Modification of the glycosylation signal of the herpes simplex virus type 1 glycopeptide gC-1 by mutation, increased the content of O-linked glycans but little effect on N-glycans.

TOF (Mårdberg, et al., 2004) Human CSGalNAcT-1 and CSGalNAcT-2 CSGalNAcT-1 is involved in the initiation of chondroitin sulfate synthesis. CSGalNAcT-2 participates mainly in elongation.

TOF (Sato, et al., 2003b) Human, GalNAc-T1, -T2, -T3, -T4, -T6, -T9 Enzymes have different substrate specificities but GalNAc-T2 was found to transfer GalNAc residues to the same five positions of the IgA1 hinge region in B cells.

R-TOF (CHCA) (Iwasaki, et al., 2003) Human, pp-GalNAc-T13 (neuroblastoma cell) Transfers GalNAc to several mucin-derived peptides including Muc5Ac and Muc7. TOF (DHB), glycopeptides (Zhang, et al., 2003e) Human, pp-GalNAc-T14 Transfers GalNAc to Muc2, Muc5Ac, Muc7 and Muc13. May be involved in Oglycosylation in the kidney TOF (CHCA) (Wang, et al., 2003c) Human,

Transfers GalNAc to the 4-position of GlcNAc in N-and O-linked glycans.

TOF (DHB) (Sato, et al., 2003c) Human, GalNAc-TIV Similar enzyme to human, β4GalNAc-T3. Transfers GalNAc in β1→4-linkage TOF, NMR Human,

Synthesises a unique carbohydrate structure, GalNAc-β-(1→3)-GlcNAc.

TOF, NMR Human, pp-GalNAc-T15 Transfers up to seven GalNAc moieties onto the Muc5AC peptide whereas the T2 enzyme only transfers five. TOF (CHCA), LC, glycopeptides Other transferases α-(1→2)-L-Gal-T from Helix pomatia Transfers an L-fucose residue to terminal D-galactopyranosyl residues, has been found to be specific for galactose with β-linkages. . R-TOF (DHB), HPAEC (Scheppokat, et al., 2003b) B4-Gal-T, CHO Lec19 cells Resistance to plant lectins such as ricin and abrin that bind galactose shown, in these cells, to be due to a reduced ability of the cells to transfer galactose to the N-linked glycans. TOF (DHB, neutral, THAP, acidic) (Lee, et al., 2003c) LpcC (Rhizobium leguminosarum) Adds a mannose unit to the inner Kdo moiety of the LPS precursor, Kdo2-lipid IVA.

TOF (ATT, ammonium citrate) (Kanipes, et al., 2003b) PimF (Mycobacterium marinum) First analysis of a Man-T involved in the later stages of phosphatidylinositol mannoside synthesis.

R-TOF, PSD (Alexander, et al., 2004) Membrane bound Gal-T, Glycine Max pectin Gal-T involved in biosynthesis of β-(1→4)-galactan chains of pectin. Gal transfer to unmodified pectic galactan (Mr>150,000) low but increased after partial acid hydrolysis. TOF (DHB) HPLC, GC/MS (Konishi, et al., 2004) AknK (L-2-deoxyfucosyltransferase) Identified as the enzyme that adds L-2-deoxyfucose as the second sugar in the carbohydrate of the antitumor drug aclacinomycin A produced by Streptomyces galilaeus.

TOF, LC/MS, NMR (Lu, et al., 2004b) Dermatan sulfate biosynthesis Clarification of controversy over whether 4-sulfation in dermatan sulfate occurs on GalNAc before epimerization of neighbouring GlcA to iduronic acid or afterwards L-TOF (DHB), HPLC (Mikami, et al., 2003) Glycosidases β-N-Acetylhexosaminidases Enzymes hydrolyse and transglycosylate β-GlcNAc and β-GalNAc moieties suggesting that they are single enzymes having affinity for both substrates. Studies in about 60 fungi TOF (Weignerová, et al., 2003) α-Amylase (Bacillus licheniformis and human saliva)

Aspergillus niger pectinase An isoenzyme that cleaves both GlcNAc-GlcN and GlcN-GlcN linkages thus offering a replacement for expensive chitinase and chitosanase currently used for this purpose.

TOF (DHB) (Kittur, et al., 2003) Endo-β-N-GlcNAc-ase from

Mucor hiemalis (endo-M) Enzyme expressed in Candida boidinni can transglycosylate a disialo-biantennary glycan in media containing 30% acetone, DMSO or MeOH in higher yield than aqueous solution.

TOF (THAP), HPLC Endo-β-N-GlcNAc-ase from

Mucor hiemalis A variety of sugar derivatives modified at C-1 or C-2 can be used as acceptors for this enzyme.

TOF (DHB, THAP), HPLC Endo-β-Gal-ase from Bacteroides fragillis Glycolysis of the Tamm-Horsfall glycoprotein releases two sugars, measurement of which in identical twins indicates closely-controlled glycosylation in the two individuals. TOF (CHCA), glycans (Rohfritsch, et al., 2004) Polygalacturonases from Trichoderma reesei Study of substrate specificity; the random pattern for pectin cleavage showed that the enzymes were exo-polygalacturonases.

TOF (THAP/nitrocellulose) (Mohamed, et al., 2003) Xyloglucanases Xyloglucanases from Aspergillus japonicus, Chrysosporium lucknowense, Trichoderma reesei. A. japonicus displayed endo-type attack whereas other two enzymes exo-type.

TOF (Grishutin, et al., 2004) Other enzymes acting on carbohydrates

Chitinases, Chit33 and Chit42 Purified from the filamentous fungus Trichoderma harzianum and their hydrolysis products characterized TOF (DHB) (Boer, et al., 2004) GDP-6-deoxy-D-talose synthetase Identification of the enzyme that converts GDP-4-keto-6-deoxy-D-mannose into GDP-6deoxy-D-talose in the Gram-negative bacterium Actinobaccillus actinomycetemcomitan.

R-TOF (THAP, NH 4 citrate) (Mäki, et al., 2003) Heparin/Heparan 2-O-sulfatase Enzyme from Flavobacterium heparinum active site and saccharide substrate specificity MALDI (CHCA) (Raman, et al., 2003) Lipid A 1-phosphatase from Rhizobium leguminosarum r  a  h  c  l  a  c  i  m  e  h  c  o  i  b  d  n  a  g  n  i  n  o  l  c  ,  n  o  i  s  s  e  r  p x E (Karbarz, et al., 2003) LpxA (Mesorhizobium loti and Leptospira interrogan) Mechanism of action in formation of lipid A with four N-linked acyl groups. Enzyme does not acylate UDP-GlcNAc but requires UDP-GlcNAc3N TOF (Sweet, et al., 2004) Pectin methylesterase from Aspergillus niger Enzyme prefers oligogalacturonides with one or two non-esterified galacturonic acids in its active cleft rather than fully methylated acids.

TOF (THAP/nitrocell) Potato D enzyme Catalyzes the cyclization of amylose to produce cycloamylose TOF (Takaha, et al., 2003) Quinone-dependent pyranose dehydrogenase Products formed by enzyme extracted from basidiomycete fungi studied with lactose as substrate. The 2-oxo-product characterized as its N,N-diphenylhydrazone derivative.

MALDI-TOF, NMR. (Volc, et al., 2004) 

PNGase F R-TOF (s-DHB), Glycans P. pastoris engineered to produce complex glycans (Bobrowicz, et al., 2004) Angiogenic vascular growth factor VEGF 121 (Human)

Pepsin, PNGase A TOF (DHB) Genes encoding the enzymes for (1→3)-fucose and 1→2-xylose disrupted. Did not affect plan to be produced without the immunogenic sugars. (Koprivova, et al., 2004) Anti-rabies monoclonal antibody Tobacco PNGase (In gel) R-TOF (DHB), Ion-trap/TOF, Glycans Structural characterization (high-mannose, complex). Glycosylation did not affect efficacy (Ko, et al., 2003) Avidin (chicken)

PNGase A R-TOF (DHB), Glycans (Reduced, Per-Me) For detection of immunogenic core (1→3)-fucose and core xylose (Bardor, et al., 2003a) Bile salt-stimulated (Trimble, et al., 2004) C1 Inhibitor (Human)

PNGase F R-TOF (DHB (+ve), THAP/NH 4 citrate (-ve)), Glycans Structural determination of glycans expressed in milk (Koles, et al., 2004a , Koles, et al., 2004b C5-1 Antibody Alfalfa PNGase A R-TOF, PSD (DHB, CHCA), glycans (Per-Me) Structural determination, Plant-type, complex (Bardor, et al., 2003b) C5-1 Antibody (Human) Alfalfa PNGase A Pepsin R-TOF (DHB, CHCA), PSD, (glycopeptides), Glycans (Reduced, per-Me) Alfalfa plants able to synthesise IgG with humantype glycans (Bardor, et al., 2003b) Chorionic gonadotrophin

Hydrazine R-TOF(DHB, CMBT), ESI, Ion trap. Glycans (2-AB) Structural determination of complex glycans and method development. (Gervais, et al., 2003) Dac g 5 and proDer p 1

Trypsin TOF (CHCA) Presence of Man can have an adverse effect on the specificity of diagnostic tests for autoimmune and allergic diseases that are based on immunoassays.

(van Oort, et al.,

Endogenous glycoproteins

Pepsin, PNGase A TOF (DHB) Enzymes adding potentially antigenic β (1→2)-Xylose and α (1→3)-fucose removed (Strasser, et al., 2004b) Erythropoietin CHO cells PNGase F TOF (DHB), Glycans (4-AA) Structural determination under different culture conditions. Fully sialylated tetra-antennary glycan is the major determinant of biological activity (Yuen, et al., 2003) Erythropoeitin CHO cells -L-TOF (Sinapinic acid, DHB, HPA, ferulic acid), ESI, CE, glycoproteins Characterization of intact glycoprotein by several methods (Sanz-Nebot, et al., 2003) Follicle stimulating hormone CHO cells Hydrazine R-TOF (DHB, CMBT), ESI, Ion trap, Glycans (2-AB) Structural determination of complex glycans and method development (Gervais, et al., 2003) into yeast. Pronase glycotetrapeptide from bovine fibrin to assay enzyme activity. (Bencúrová, et al., 2003) GM2-activator protein

PNGase F TOF (DHB), ESI-TOF, NMR Structural determination (Wendeler, et al., 2004b) Hyaluronan synthase (human) E. coli -L-TOF (DHB) Expression of catalytic region of human hyaluronan synthase (HAS2) in E. coli shown to synthesise a mixture of HA oligomers from 8-to 16-mer. (Hoshi, et al., 2004) IgG ( (Sriraman, et al., 2004) IgG 1 (Human) Rat hybridoma or CHO Hydrazine MALDI, HPLC, Glycans (2-AP), Absence of fucose but not presence of galactose or bisect enhances cellular toxicity (Shinkawa, et al., 2003) IgG 3 M o u s e Hydrazine, PNGase F R-TOF (+ve) (DHB), ESI, MS/MS, Glycans Effect of buffer and pH on production. (Müthing, et al., 2003) IgG 4 (Human)

Trypsin TOF (CHCA), Glycopeptides Glycosylation (high-mannose, truncated complex) not significantly affected by anti-mitotic agent (Tait, et al., 2004) KDEL-tagged cPIPP antibody Structural characterization (Fujiyama, et al., 2004) Lecithin:cholesterol acyltransferase (Lane, et al., 2004) Luteinizing hormone (Human)

Hydrazine R -TOF (DHB, CMBT), ESI, Ion trap, Glycans (2-AB) Structural determination of complex glycans and method development (Gervais, et al., 2003) MFE-CP

Trypsin R-TOF, LC/MS, CID, PSD, Glycopeptides Structural characterization of glycoprotein (highmannose, Asn-442, 484) used to target cancer cells (Medzihradszky, et al., 2004) (Continued )

Onconase

Endo-H TOF, glycoprotein Cancer chemotherapeutic agent. Structures of high mannose glycans (Man 9-16 ) from mass of glycoprotein Ovotransferrin (Chicken)

Endo H TOF (Sinapinic acid), Glycoprotein, Glycan mass by difference Structural characterization (Mizutani, et al., 2004) Placental alkaline phosphatase (Human)

PNGase F TOF (DHB), Glycans (2-AB) Presence of silkworm hemolymph improvesNglycosylation, High mannose (Joosten, Park & Shuler, 2003a) Plasminogen Kringle

PNGase F TOF (s-DHB) Glycosylation machinery re-engineered to perform sequential glycosylation reactions that mimic Nglycan processing in humans. Plasminogen Kringle

PNGase F TOF Yeast engineered to produce GlcNAc 2 Man 3 GlcNAc 2 Plasminogen Kringle

PNGase F TOF PpALG3 gene that encodes for Dol-P-Man:-Man 5 GlcNAc 2 -PP-Dol Man-T deleted to prevent further mannose addition. Incorporation of a -(1 4)-Gal-T gave human-type glycans. (Bobrowicz, et al., 2004) Procarboxypeptidase Y Pichia pastoris -TOF (Sinapinic acid), Glycoprotein Replacement of all Asn of N-glycosylation sites by Ala removed N-glycosylation. Residual glucose thought to be O-linked. (Maeda, et al., 2004) Soluble human complement receptor type 1 (sCR1)

PNGase F TOF (DHB), Glycans (2-AA) Glycan remodelling using the soluble glycosyltransferases, -(2 3)-silyltransferase to introduce silyl Lewis X moieties. (Thomas, et al., 2004a) Transferrin Insect cell line PNGase A Trypsin + Chymotrypsin TOF (DHB), Glycans (2-AP) Trichoplusia ni insect cells engineered to produce biantennary glycans (Tomiya, et al., 2003) Transferrin (Human)

Lymantria dispar (gypsy moth) PNGase A L-TOF (DHB), Glycans (2-AP) Attempt to find a better expression system than those currently used. Absence of core (1 3)fucosylation, a normally common motif in insects. (Choi, et al., 2003b) Transferrin (human)

Pepsin, PNGase A L-TOF, Q-TOF (CHCA), Glycans, glycopeptides To study specificity of IgG and IgE antibodies against (1 3)-fucose and (1 2)-xylose epitopes. Binding greatest for fucosylated glycans. (Bencúrová, et al., 2004) Vascular growth factor VEGF (1 2)-xylose enzymes (Koprivova, et al., 2004 , Koprivova, 2003 #2924) Glucose-based glycoprobes Ident. of binding sites in Na/D-glucose co-transporter protein L-TOF (Sinapinic) (Tyagi & Kinne, 2003) Monosaccharide amides Ru(III)-Promoted amide formation from azides and thioacids, which do not form amides at room temp. in the absence of Ru FT-ICR (Fazio & Wong, 2003) Quaternary ammonium salts of ribitol Salts from five aromatic amines TOF (CHCA) Oligosaccharides N-Acetylglucosaminobioses By reverse hydrolysis using fungal N-acetylhexosaminidases TOF (Rauvolfová, et al., 2004b) Arabinogalactosyl nonasaccharides β-(1→6)-D-Galp backbone, α-(1→3)-linked L-Araf side-chain TOF (DHB) (Li & Kong, 2004) (Argyropoulos & Sarli, 2004) Biotinylated saccharides As part of avidin/biotinylated drug delivery system TOF (Ouchi, et al., 2004) 6-Branched cyclo-(1→3)-glucohexose Cyclisation of branched glucohexaose TOF (Wu & Kong, 2004b) 2,6-Branched galacto-oligosaccharides Synth. of tetra-and hexa-saccharides related to arabinogalactans TOF (Ning, Yi & Yao, 2003b) Carbohydrate-modified poly(di-Me-siloxane)s Linear dimethylsiloxanes capped with various monosaccharides TOF (Henkensmeier, et al., 2004) 6-O-carboxymethylated chitotetraose Selective glycosidation with chitinase catalysts TOF (Ochiai, Ohmae & Kobayashi, 2004b) Chitobiose analogues Sugar oxazolines glycosylated with chitinase from Bacillus sp.

Nafion plate (Ochiai, Ohmae & Kobayashi, 2004a) Chitooligosaccharides As standards for diffusion-ordered spectroscopy TOF (Groves, et al., 2004) Chitooligosaccharides Synthesis from chitosan by enzymatic hydrolysis with chitinase TOF Chitooligosaccharides DP 2-6 Use of pectinase from Aspergillus niger TOF, FAB (Kittur, et al., 2003) Chititriose nicotinic amide For conformational studies. Inhibitor of chininase hevamine MALDI (Germer, et al., 2003) C (Galan, et al., 2003) Cyclosophoraoses of Rhizobium meliloti 2011 Enzymatic synthesis. As additive for enantioseparation in CE TOF Deoxy phospha sugar-sugar disaccharides Phospha analogues of normal sugar disaccharides TOF (CHCA) (Haritha, et al., 2004) Fucosyl-lactoses Specific Fuc addn. after lipase-catalysed regioselective acylation TOF (Rencurosi, et al., 2003) Fucosyl pentasaccharide (sulfated fucan repeat) Convergent "2+3" synthetic strategy TOF (DHB) (Hua, et al., 2004a) Fucosylated trisaccharides

Study of the specificity of the α-(1→2)-L-galactosyltransferase from Helix pomatia. Specific for D-Galp β-linkages TOF (DHB) (Scheppokat, et al., 2003b) Fucosylated trisaccharides Synthesis with α(1→2)-L-galactosyltransferase from Helix pomatia.

TOF (DHB) (Scheppokat, Bretting & Thiem, 2003a) Fucosylated trisaccharides (lactose)

Random fucosylation to construct library TOF (Meng, et al., 2004) Gal-β-(1-3)-GlcNAc Use of an anomeric fluorous silyl protecting group TOF (Manzoni, 2003) Galactans Biosynthesis by use of galactosyltransferase from radish (Raphanus sativus L.) seedlings TOF (ABEE derivs.) (Kato, et al., 2003) Galactose-containing oligosaccharides β-Gal-T from bovine testes to synth. novel food components TOF (DHB) (Schröder, et al., 2004) β-(1→6)-D-Galactosylated oligosaccharides Use of β-(1→6)-D-Gal-transferase from Helix Pomatia TOF (Scheppokat, et al., 2004) Galactosylated tri-and tetrasaccharides Regioselective synth. β-galactosidase from Bacillus circulans TOF (DHB) (Farkas, et al., 2003) , et al., 2004d) β-D-Glucosamine-containing nonasaccharide Chemical synthesis with isopropyl thioglycosides TOF (CHCA) β-D-Glucosamine-containing oligosaccharides Chemical synthesis with isopropyl thioglycosides TOF (CHCA) (Yang, Hua & Du, 2003) Glucosylated isokestose and nystose Enzymatic (kojibiose phosphorylase from Thermoanaerobacter brockii to synthesise five novel oligosaccharides TOF Glycosaminotrioses With GalNAc or ManNAc termini. To study binding to hevein from Hevea braziliensis TOF (CHCA) (Aboitiz, et al., 2004) Imino sugar scaffolds For the generation of glycosidase inhibitor libraries TOF (DHB) (La Ferla, et al., 2004) Kanamycin analogues Chemical synthesis and antibacterial evaluation MALDI (Li, et al., 2004b) Lacto-N-neotetraose Dendrimeric polyethylene glycol supported synthesis TOF Laminarin hexasaccharide 4,6-O-benzylidenated acceptor to avoid generation of α−isomer in attempted β-D-(1→3) glycosylation under standard condits. TOF (CHCA) (He, Gu & Du, 2003) Lepidimoide Unsaturated disaccharide (4-deoxy-β-L-threo-hex-4enopyranosyl-(1→2)-L-Rhap) from okra mucilage TOF (Co powder) )

Chemical. Use of anomeric fluorous silyl protecting group TOF (Manzoni & Castelli, 2004) Maltooligosyl fructofuranosides Immobilized cyclodextrin glucosyltransferase catalyst TOF (DHB) Maltosyl-erythritol Transglycosylation product of erythritol by maltogenic amylase TOF (CHCA) (Yoon, et al., 2003) α-(1→3)-linked manno-hexaose and -octaose α-(1-3)-linked di-and tetra-saccharide donor + tetrasaccharide TOF (Chen & Kong, 2002) Mannosides Synthesis by double differential glycosylation by lanthanide triflates TOF i In situ method for identification of novel α-amylase inhibitors R-TOF N- (methyl-2,3,4-tri-O-acetyl-6-deoxy-α-andβ-D-glucopyranoside-6-yl (Remenyik, et al., 2003) Oligosaccharides (di-, tri-and tetrasaccharides) Solid-phase synthesis with azidoglucose as a glycosyl acceptor TOF (DHB) (Wu & Schmidt, 2004) Oligosaccharides (general) Polymer -resin hybrid capture-release strategy for rapid oligosaccharide construction TOF (Hanashima, Manabe & Ito, 2003) Oligosaccharides (general) Reiterative intramolecular glycosylation by use of a rigid spacer TOF (DHB) (Paul, Müller & Schmidt, 2003) Oligosaccharides (rhamnan backbone) Synthesis of oligosaccharides consisting of α-(1→2)-and α-(1→3)-linked rhamnan backbones and GlcNAc side chains TOF (Zhang & Kong, 2003d) n-Pentenyl arabinofuranosyl donors Synthesis of little-studied donors for furanosides TOF N(OCH 3 )-linked disaccharide analogues Carbohydrate mimics resistant to hydrolysis by glycosidases TOF (DHB) Non-reducing oligosaccharides By reverse hydrolysis using fungal N-acetylhexosaminidases TOF (Rauvolfová, et al., 2004a) Silyl Lewis x and silyl Lewis a precursors Determination of recombinant (2→3)-α-silyltransferase TOF (DHB) (Ivannikova, et al., 2003) Sucrose laurate Enzymatic (alkaline protease from Bacillus pseudofirmus AL-89 TOF (CHCA) (Pedersen, et al., 2003) 6-Sulfo-GlcNAc-β-(1→3)-Gal-β-(1→4)-Glc Product of 6-sulfo-transferase produced in baculovirus system TOF (DHB) (El-Fasakhany, et al., 2003) (Renaudie, et al., 2004) (Prosperi, et al., 2004) Various oligosaccharides Automated solid-phase synthesiser. 20 times faster than solution TOF (DHB) (Palmacci, et al., 2003) Various oligosaccharides

Formylacetal (CH 2 ) as novel linker on soluble-polymer support TOF (Oikawa, et al., 2004) Various oligosaccharides Use of a novel hexakisfluorous butanoyl support TOF (CHCA), ESI (Goto, et al., 2004) Various oligosaccharides

Use of fluorous-tagged saccharide primers TOF (DHB) (Kasuya, et al., 2004) Various oligosaccharides Acetyl esterase from Trichoderma reesei RUT-C30 found able to transglycosylate carbohydrates in organic solvents TOF (DHB) (Kremnický, Mastihuba & Côté, 2004) Polysaccharides

C h e m i c a l ( n-pentenyl-arabinofuranosyl donor) TOF Arabinogalactans from Echinacea purpurea Synthesis of double-branched isomeric nonasaccharides TOF (Csávás, et al., 2003) C TOF (Faijes, et al., 2004) Lepidimoic acid from Hibiscus esculentus Chemical degradation of okra TOF (Co powder) (Hirose, et al., 2003) Maltobi-and -triose 2,4-dinitrophenyl-derivs.

Substrates for human pancreatic α-amylase. Kinetic analysis R-TOF Mannan of Candida kefyr IFO 0586, cell wall First synthesis of penta-and deca-saccharide repeats TOF (CHCA) (Xing & Ning, 2003) , et al., 2003) 

Chondroitin sulfate E hexasaccharide Synthesis from β-D-GalNAc-(1→4)-β-D-GlcA TOF (Tamura & Tokuyoshi, 2004) Heparan sulfate fragments Use of 6 orthogonal protecting groups to synth. 20 disaccharides TOF (-) (DHB) (Prabhu, Venot & Boons, 2003) Heparin-like fragments Synthesis of hexa-and octasaccharides on a solid support TOF (Ojeda, et al., 2004) Heparin fragments

Chemical synthesis via regio-and stereo-selective glycosylation FTMS Heparin-like hexasaccharides Synthesis by convergent block strategy TOF (DHB) (Lucas, et al., 2003) Isotope-labelled hyaluronan oligomers 15 N-, 13 C-labels for NMR studies TOF (DHB) (Blundell, et al., 2004) Monodisperse hyaluronan oligosaccharides Chemoenzymatic synthesis with immobilized mutant enzymes R-TOF (ATT) (-) (DeAngelis, Oatman & Gay, 2003) Carbohydrates from algae

Fucoidan repeat from brown algae Chemical synthesis. Good antitumor activity TOF (DHB) (Hua, Gu & Du, 2004b) 

Disaccharide fragments of LPS from several species Jones oxidation of allyl glycosides TOF (CHCA) (Madaj, Jankowska & Wisniewski, 2004) Lipid A from Chlamydia trachomatis Chemical synthesis and purity assessment TOF (DHB) (Zamyatina, et al., 2004) Mycobacterium tuberculosis phosphoinoside intermediate Use of regioselective mannosylations MALDI (Jayaprakash, Lu & Fraser-Reid, 2004) O-Chain from Campylobacter jejuni Trisacch. from GlcNPhth-(1→3)-Gal + allyl 6-deoxy-altroHep TOF (2,4-DHB) (Yoon, et al., 2004b) O-Chain repeat from

Helicobacter pylori Chemical synthesis of unique trisaccharide from Danish strains TOF O-Chain repeat from Shigella flexneri Chemical synthesis of tetra-and two pentasaccharide fragments - (Mulard & Guerreiro, 2004) Capsular polysaccharide from (Alpe, Oscarson & Svahnberg, 2003) EPS repeat from Cryptococcus neoformans Chemical synthesis of repeating unit TOF (Zhang & Kong, 2003a) Repeat (Zhang & Kong, 2003b) Lipoarabinomannan from Rhodococcus equi Chemical synth of pentasaccharide repeat from equine pathogen TOF (CHCA) (Ma, Zhang & Kong, 2004) 

Chemical plus enzymatic synthesis TOF (DHB) (Furuike, et al., 2003) Biotinylated, sialylated biantennary glycan Enzymatic synthesis with endo-M TOF (DHB) (Mori, et al., 2004) High-mannose type attached to ethylene glycol As microarrays for gp-120 (from HIV) interaction studies TOF (Adams, et al., 2004) Hybrid-type, bisected glycan Transglycosylation with endo-β-N-acetylglucosaminidase TOF (THAP) α-D-Man-(1→4)-β-D-GlcNAc-(1→4)-D-

Synthesis of unnatural α-D-mannose isomer TOF (DHB) α-(2→3)-sialic acid-containing N-glycans

Use of α-(2→3)-sialyltransferase TOF (Fukae, et al., 2004 (Hernández, et al., 2004) (Xian, et al., 2004) Complex N-glycans-glycopeptides Chemoenzymic synth. of sugar-Asn. solid-phase synth. of g-pep TOF Core II O-linked glycopeptide Solid phase plus enzymatic synthesis TOF (DHB) (Takano, et al., 2003) Core II O-linked glycopeptide Solid-phase synthesis plus enzymatic sialylation TOF (Takano, et al., 2004) Core II sialyl Lewis-X O-glycopeptide (Gallego, et al., 2003) Cyclic glycopeptides Macrolactamization of glycosylated peptide thioesters MALDI Eel calcitonin plus N-glycans Transglycosylation with GlcNAc-ase from Mucor hiemalis TOF (CHCA) (Haneda, et al., 2004) Emmprin with N-linked core pentasaccharide Chemical, solid phase.

N-glycosylated Ig domain TOF Epidermal growth factor-like domain of blood coagulation factor IX carrying Xyl-Glc Solid-phase synthesis. Mild conditions enabled protecting groups on the sugar to be omitted TOF (Kitamura, et al., 2004) Epithelial cadherin glycopeptides Solid-phase synthesis. Fmoc chemistry TOF (Reipen & Kunz, 2003) Epithelial mucin MUC4 Synthesis of tumor-associated glycopeptide by solid-phase synthesis TOF (Brocke & Kunz, 2003 , Brocke & Kunz, 2004 Galactosylated 5-hydroxylysine mimetics For incorporation into peptides such as type II collagen TOF (Marin, et al., 2004) (Sato, et al., 2004b) Glycopeptides (of cell-surface glycoproteins) Use of (2-Ph-2-TMS)ethyl-(PTMSEL) as a fluoride-sensitive anchor for solid-phase synthesis TOF (Wagner, Dziadek & Kunz, 2003) Glycopeptides Study of glycosylation catalysed by PNGase F in reverse rn.

(CHCA) (Jeong, Lee & Park, 2004) Glycopeptide mimetics Incorporation of unnatural (keto) amino acids in peptide chain FTICR (Liu, et al., 2003a) Glycopeptide from collagen Chemical synthesis. Helical models incorporating collagen sequences for receptor binding studies TOF (DHB, sinapinic acid) (Lauer-Fields, et al., 2003) Gly-Pro-Thr(β-Gal) 10 Carbohydrate stabilizes triple helix of collagen by H-bonds TOF (Bann, Bächinger & Peyton, 2003) Heterocyclic-tethered C-glycosyl amino acids , et al., 2003) Interleukin-1α plus Neu5Ac-Gal Coupling by acyl azide, 2.5 mols/mol of protein TOF (Ootsubo, et al., 2004) β-Lactoglobulin Glycosylation by heating with Glc or Fru for thermal stability TOF (CHCA) (Broersen, et al., 2004) (Halkes, et al., 2003) S-Linked glycopeptides Wo-phase system to avoid use of strong base MALDI (Zhu & Schmidt, 2003) S-Linked glycopeptide derived from human Tamm-Horsfall glycoprotein Direct base-catalyzed S-glycosylation of cysteine and Homocysteine-containing peptide with O-Ac protected bromides MALDI (Zhu, Haag & Schmidt, 2004c) Sperm CD52 Fmoc chemistry to build peptide chain with glycan-Asn TOF (Shao, Xue & Guo, 2004) Glycosides Acylated ginsenosides Regioselective acylation in organic solvents with vinyl acetate and Candida antarctica lipase TOF (CHCA) (Teng, et al., 2003 , Teng, et al., 2004 1-Adamantylmethyl glycosides Single-step synthesis from per-Ac-monosaccharides. Binding to cyclodextrins depends on pyrenose stereochemistry TOF (Charbonnier & Penadés, 2004) β-D-Aminoglycosides Use of 2-nitro-thioglycoside donors TOF (DHB) (Barroca & Schmidt, 2004) Diadzein glycosides

Transglycosylation with Thermotoga maritima maltosyltransferase to increase solubility L-TOF (DHB) Dihydrodiosgenin glycosides As mimetics of bidesmosidic steroidal saponins R-TOF (DHB) (Suhr, et al., 2003a) (Joosten, et al., 2003b) Glycosyl glycerol Transglycosylation by cyclodextrin glucanotransferase TOF (CHCA) (Nakano, et al., 2003a) (Continued ) (Dondoni & Perrone, 2003) Lipo-chitooligosaccharide nodulation factors Two-step chemical and biotechnology synthesis TOF p-Methoxyphenyl β-lactosides with 3'supfonic acid To find carbohydrate ligand(s) which can inhibit adhesion of Helicobacter pylori and gastrointestinal epithelial cells.

MALDI (Borbás, et al., 2004) OSW-1 Analogues (cholestanes saponins) Mod. of steroid side-chain. No change in anti-tumor action.

MALDI (Deng, et al., 2004) Rhamnosylated diosgenyl glucosides As mimetics of cytostatic steroidal saponins from Ornithogalum saundersiae and Galtonia candicans R-TOF (DHB) (Suhr, et al., 2003b) ) , et al., 2003) 3'-Substituted p-methoxyphenyl β-lactosides To find ligand(s) which can inhibit the adhesion between

Helicobacter pylori and gastrointestinal epithelial cells.

MALDI (Borbás, et al., 2004) Glycosphingolipids

Reaction of thioglycosides with benzoyl ceramide TOF (Yamamura, et al., 2004) 

Mannobiose-linked phosphoethanolamine Chemical synthesis of GPI anchor intermediate TOF (DHB) Glycolipids Dolichylphosphomannose analogues Potential inhibitors of Man-T in endoplasmic reticulum MALDI (DHB) (Kulesza, et al., 2004) Glycolipids (general) Ag triflate as alternative to TMS-triflate for sensitive rns.

TOF (CHCA) Glycolipids with partially F-alkyl chains (Stadelmaier, et al., 2003) Maltosyl erythritol Transglycosylation with amylase (Bacillus stearothermophilus) TOF (CHCA) (Yoon, et al., 2003) 6-O-Sulfo Lewis X neo-glycolipid Containing lactamized neuraminic acid TOF (DHB) Cyclodextrins Acetylated α, β and γ cyclodextrins Chemical synthesis. Properties in dense CO 2 TOF (Potluri, et al., 2003) Allylated cyclodextrin (6-NH-(CH 2 ) 3 -Si(OEt) 3 ) 7 substituents. As chiral HPLC phase.

TOF (DHB) (Lai & Ng, 2004) Amphillic cyclodextrins Novel monosaccharide carriers through bulk liquid membranes TOF (Kida, et al., 2004) Amphiphilic CDs fully substd. on primary face Chemical and enzymatic synthesis. 6-Substituted CDs TOF (Sallas, Niikura & Nishimura, 2004) Bis-β-Cyclodextrin/ pyromellitic acid 2,5-diamidate Chemical. Improved binding of guest dyestuff result of double cavity.

TOF (Liu, et al., 2003b) Cationic cyclodextrins 6-NHPy, 6-Me-imidazole, 6-Bu-imidazole, 6-NH-C 2 H 4 OCH 3 , NH 2 . Plasmid complexes for transfection MALDI (Cryan, et al., 2004) 6-N-Carboxyalkyl (-NH-CO(CH 2 ) 4, 5 or 10 COOH) 7 substituents, Enzyme promoter MALDI 6-O-Carboxymethyl (Per-Me) (CH 2 COOH) 7 , (Me) 14 substituents. CEC R-TOF (+) (THAP) (Culha, et al., 2004) Cyclodextrin dimer For transport of saccharides through a liquid membrane TOF (Ikeda, Matsuhisa & Ueno, 2003) Cyclodextrins with acetone bridge Epoxidation catalysts. Ozone, catalysed epoxidation of alkenes TOF (CHCA) (Rousseau, et al., 2004) (Teranishi, 2003) Cyclodextrins with methacryl moieties Characterization, copolymerization with 1-vinyl-2-pyrrolidone TOF (DHB) (Janus, et al., 2003) CDs with one Dithienylethene-tethered β-cyclodextrin dimers Photocontrolled release and uptake of a porphyrin guest TOF (Mulder, et al., 2004) Fluorescent cyclodextrin/peptide hybrids with macrocyclic metal complex ZnII-cyclen as a ligand site and β-CD is a receptor site for guest molecules, while the dansyl unit acts as a fluorescent probe.

TOF (Furukawa, Mihara & Ueno, 2003) Glycosylated β-cyclodextrin (β-Gal) 1,2 , (Lac) 1,2 , β-(Gal-Lac) 1,2 subs. Molecular recognition R-TOF (+) (DHB) (Ikuta, et al., 2004) Mannoside substituted (Harabagiu, et al., 2004) Pseudopolyrotaxanes + lactose-substituted CDs

Receptor binding studies (Lac) 6 (Lac) 7 substituents FT-ICR (DHB) (Nelson & Stoddart, 2003) Pyridylmethyl-per-polyethyleneglycol-β-CD Synthesis of model hemoprotein. CH 2 -Py, -(C 2 H 4 O) 3 -CH 3 subs.

TOF (Zhou & Groves, 2003) Na dodecyl sulfate + CD-based nanotubes Study of binding in formation of inclusion complexes TOF (Kalashnikov, et al., 2004) , et al., 2004b) β-Alanine-based glycoclusters β-Alanine polypeptide plus galactose TOF (Sato, Hada & Takeda, 2003a) Carbohydrate amphiphiles Hydrazone ligation with lipophilic glyoxylyl acid derivatives TOF (Grandjean, et al., 2004) Carbohydrate-centred glycoclusters Penta-and 15-valent mannosides, glucose centre TOF (DHB) (Köhn, et al., 2004) Carbosilane dendrimers Tetravalent glycodendrimers. Two new synthesic methods TOF (2,4-DHB) (Choudhury, et al., 2003) i L ( , et al., 2003) GalNAc-Containing biotinylated clusters Lgands of asialoglycoprotein receptor for targeted gene delivery TOF (DHB,THAP) (Westerlind, et al., 2004) Glycerol and glycol oligomers with mannose To study multivalent binding with proteins TOF (DHB) (Continued ) (Oshovsky, et al., 2004) Glycoclusters from silsesquioxanes Prepared via thiol-radical addition reaction TOF (Gao, et al., 2004) o , et al., 2004) Peptide dendrimers based on β-cyclodextrin Thiol link to 6-position of glucose residues TOF (Muhanna, et al., 2003) Poly(n-butyl-2-cyanoacrylate)/dextran nanoparticles Comparison of methods for nanoparticle characterization TOF (DHB) (Weyermann, et al., 2004) Polyphenylene-based glycodendrimers Synthesis of glycodendrimers with sugars within scaffold TOF (Sakamoto & Müllen, 2004) Porphyrin-containing glycodendrimers With four and twelve derivatized glucose residues TOF (CHCA) (Ballardini, et al., 2003) Schizophyllan with β-lactoside and αmannoside appendages NaIO 4 oxidation followed by reductive amination with aminoethyl-sugars TOF Spherical and hemispherical poly(ornithine) glycodendrimers Maltose or lactose. Up to 32 arms TOF (DHB) (Baigude, et al., 2004a ) (Baigude, et al., 2003) TEMPO-functionalized PAMAM dendrimers

With increasing mannose loadings TOF (3-IAA) (Samuelson, et al., 2004) Thioglycosylated cationic porphyrins Potential photodynamic drugs for colorectal adenocarcinoma MALDI (Ahmed, et al., 2004) tri ( 

Bisected biantennary glycans plus BSA hemoenzymatic synth. Bisect modulates ligand properties TOF (DHB) (André, et al., 2004) Core of E. coli R4 plus tetanus toxoid Synthesis via reductive amination TOF (THAP) (Lukasiewicz, et al., 2003) Fluorescent saccharide biosensors plus Con-A Photoaffinity labelling to concanavalin A TOF (DHB, CHCA, sinapinic) (Nagase, et al., 2003) GD 3 , GQ 1b and GM 2 epitopes plus BSA For oligosaccharide-specific immunoadsorption therapy of Guillian-Barré syndrome TOF (Andersen, et al., 2004) Glycopeptides from Group A Streptococcus and Shigella flexneri Y plus BSA or TT Diethyl squarate coupling TOF (DHB, sinapinic acid) (Hossany, et al., 2004) LDNT, LDNFP and LNFPIII plus BSA Study of targets for immune intervention in schistosomiasia TOF Lewis b hexasaccharide plus HSA Linked with disuccinimidyl suberate spacer TOF (THAP) (Zhu, et al., 2004b) O-Specific polysaccharides from Vibrio cholerae plus BSA Di-to penta-saccharides linked via squaric acid diester chemistry SELDI-TOF (Saksena, et al., 2003 ) Schistosoma mansoni glycan fragments + BSA Squaric acid chemistry. For studies on antibody binding TOF Trisaccharides from Toxocara sp. plus BSA Trisaccharides bound to BSA to act as immunoreagents TOF (+ve) (DHB) (Amer, Hofinger & Kosma, 2003) Truncated complex glycans plus BSA Ninhydrin coupling of pronase glycopeptides for binding studies MALDI (van Remoortere, et al., 2003) Polymers Allylurea-carbohydrate polymers Synthesis of biodegradable material similar to thermoplastic TOF (DHB) (Olivier, et al., 2003) (1,2: (Iyer, et al., 2003) Polyacetyl containing trehalose Reaction of α,α-D-trehalose with terephthaldehyde. MW 8 kDa TOF (CHCA) (Teramoto, et al., 2004) Polymers by ring-opening metathesis From norborene-attached acetyl-protected carbohydrates TOF (IAA) Schizophyllans with β-lactoside and α-Man Synthesis by NaIO 4 oxidation followed by reductive amination TOF Antibiotics/Anticancer (Menéndez, et al., 2004) Kanamycin A-cholesterol conjugates Aminoglycoside-derived cationic lipids for gene transfection R-TOF (DHB) (Sainlos, et al., 2003) Landomycin E clusters New compounds synthesised by biosynthetic and LndGT4 gene manipulation TOF (Ostash, et al., 2004) Squaric acid amides of anthracycline antibiotics For covalent binding to biomolecules TOF (Tevyashova, et al., 2004) Vancomycin analogues One-pot synth. of modified carbohydrate after glycan removal MALDI (Ritter, et al., 2003) Miscellaneous Azido-glycoside on a sensor chip Mannose-C 12 glycoside attached by two methods TOF (Sato, et al., 2004c) Ferrocene-carbohydrate complexes Two methods to attach mono-and di-saccharides to one or both cyclopentadienyl rings. Electrochemical behaviour studied. , et al., 2003) Oligosaccharide microarrays

Assembly of sugars on polystyrene plates FT-ICR (Fazio, et al., 2004) ANALYSIS OF CARBOHYDRATES AND GLYCOCONJUGATES & the two areas that are particularly relevant for monitoring are those of glycodendrimers and glycoprotein conjugates.

Many polyfunctional compounds have been used to build the basic scaffold of these molecules. Dendrimers built on such scaffolds include those on tris-(2-aminoethyl)-amine (60) (Li et al., 2004e) , trimesic acid (61) (Nishida et al., 2003) , carbosilanes (62) , silsesquioxanes (63) (Gao et al., 2004) , cyclotriveratrylene (64) (van Ameijde & Liskamp, 2003) , tetra-aryl porphorins (65) (Ballardini et al., 2003; Ahmed et al., 2004; Laville et al., 2004) and fullerene derivatives (Isobe et al., 2003) . Further examples are in Table 11 . Poly-hydroxy compounds such as carbohydrates themselves have been used as the core for dendrimers. Thus, Wu and Kong (2004a) used ethylene glycol and glycerol to produce di-and trivalent (66) trisaccharides. Three first-order dendrimers based on a cyclodextrin core containing fourteen Val, Phe or Val-Phe residues have been synthesized. The amino acids were linked to the terminal residues of 3,3 0 -iminobispropanolamine which was coupled through the central nitrogen to the 6-iodo derivative of bcyclodextrin (Muhanna et al., 2003) . First-order dendrimers have also been synthesized by coupling 7, 14, or 21 Man-O-Ph-C:C-CH 2 -groups to b-cyclodextrin (Ortega-Caballero, Giménez-Martínez, & Vargas-Berenguel, 2003) (67) giving glycodendrimers with carbohydrate moieties both at the core and periphery. Wu and Kong (2004a) have also synthesized related compounds by cyclization of nona-and dodecasaccharides to give dendrimers containing hexa-and octa-saccharide rings. Schizophyllan ([ ! Glc-b-(1 ! 3)-(Glc-b-(1 ! 6)Glc-b-(1 ! 3)-Glc-b-(1 ! 3)-] n ) has also been used as a backbone;

b-glucoside appendages were oxidized to give aldehyde groups which were coupled to aminoethyl-b-lactoside or a-mannoside by reductive elimination . There is also a report of a glycodendrimer, based on a polyphenylene scaffold with a ring of monosaccharides between the center and the periphery (Sakamoto & Müllen, 2004) (68) . Some of the largest multivalent glycodendrimers are based around a scaffold consisting of amino acids or polyamidoamine (PAMAM). In one of the latter cases, MALDI-TOF analysis of the products gave broad peaks with masses less than those predicted and attributed to incomplete synthesis of the PAMAM scaffold. Masses ranged from 10,000 to 132,000 Da (178 mannose residues) (Woller et al., 2003) . Choudhury, Kitaoka, and Hayashi (2003) with 32 sugar units produced a spectrum (m/z 19,132) but the next generation glycodendrimer (64 sugar units, predicted mass for C 1518 H 2656 O 892 N 250 ¼ 38,661) failed to give a signal. Synthesis of large, fifth generation glycodendrimers with potentially 64 outer sugar residues (69) and sulfated analogues has been monitored by MALDI-TOF MS from THAP (positive ion) and IAA (negative ion) to reveal that approximately 50 of the potential 64 sugars were actually incorporated (Kensinger et al., 2004) . Sharp MALDI-TOF peaks from spherical oligosaccharide-b-alanine-poly(lysine) dendrimers containing 32 cellobiose (13,418 Da), maltose (13,472 Da), and lactose (13,507 Da) groups respectively, synthesized by Baigude et al. (2003) , showed complete substitution of the amino groups by carbohydrate. Differences were found between the measured and calculated masses of larger maltose-containing glycodendrimers (13 kDa) based of a poly(ornithine) scaffold with the higher experimental masses being attributed to instrumental factors. The MALDI-TOF spectrum from CHCA of a third generation maltose-proline-poly(lysine) dendrimer (4.4 kDa) synthesized by Baigude et al. (2004b) gave three peaks attributed to the [M þ H] þ , [M þ Na] þ , and [M þ K] þ ions. Attempted addition of a succinyl group to each maltose residue produced a MALDI spectrum with multiple peaks attributed to different numbers of attached succinyl groups and giving an average number of 13 attached groups. Finally a 24-mer peptide was attached to produce a potential AIDS vaccine; MALDI-TOF analysis indicated the addition of one and two such units. Self-assembling dendrimers carrying four or eight GalNAc residues have been constructed on a bipyridyl core; addition of Cu(II)SO 4 linked two bipyridyl units to give the dendrimer. It was characterized by (70) (Roy & Kim, 2003) .

Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) analysis is used in this area mainly to estimate the number of carbohydrate molecules bound to the protein.

Conjugation of the glycans is frequently by squaric acid chemistry or by reductive amination. However, Lewis b hexasaccharide has been linked to human serum albumin (HSA) with a disuccinimidyl suberate spacer. The authors found that this spacer had a similar linking efficiency to squaric acid but had the advantage of a fast incorporation of the receptor saccharide to the carrier protein . Saksena, Ma, and Kovác (2003) have used SELDI to monitor the conjugation to BSA of di-through penta-saccharides that mimic the upstream terminus of the O-specific polysaccharide of Vibrio cholerae O:1, serotype Ogawa using squaric diester chemistry. SELDI monitoring of the reaction allowed the extent of conjugation to be monitored and stopped when the desired molar hapten-BSA ratio had been reached. The accuracy of the mass measurement could be increased by using the carrier protein as the internal standard. Tri-and hexa-galacturonates have been synthesized and coupled to BSA by reductive amination; MALDI analysis gave a molecular weight of 70.996 corresponding to 3.8 mol of ligand/mol of BSA (Clausen & Madsen, 2004) . To develop antibodies to Saikosaponin A, a triterpenoid saponin from the root of Bupleuri radix, a plant used in Eastern medicine, Zhu et al. (2004b) have conjugated the saponins to BSA and determined the hapten number as 11 by MALDI from sinapinic acid. Concanavalin A has been labeled with a fluorescent probe at the active site by first binding a photolabile linker attached to mannose to the active site. The probe was then fixed to the protein by photoaffinity labeling, the mannose was removed by reduction of the disulfide link and the resulting thiol group used to attach various fluorescent reagents (Nagase et al., 2003) . MALDI from sinapinic acid was used to monitor various stages of the reaction and to demonstrate that the final addition of the fluorescent group occurred to the extent of 70-90%. Nyame et al. (2003) have synthesized di-and trisaccharides bearing the LDN and LDNF epitopes and conjugated these, together with one carrying the Lewis x epitope, to BSA. MALDI-TOF analysis showed that three to four molecules were coupled per mole of BSA. These and additional examples are listed in Table 11 .

A cyclosophoraose (cyclic-(1 ! 2)-b-D-glucan) named Cys, produced by soil micro-organisms of the genus Rhizobium, has been found to act as a catalyst for the methanolysis of several compounds such as phosphatidylcholine (Lee & Jung, 2004) . MALDI analysis of several of the reaction intermediates showed acylated cyclosophoraoses in the mass range 3,000-4,500. Carboxymethylated Cys from R. leguminosarum biovar trifolii could be used to complex and, consequently increase the aqueous solubility of hydrobenzoin and N-acetyltryptophan (Lee et al., 2004c) .

A few additional papers documenting the use of MALDI analysis for carbohydrate analysis that do not fit the above categories, have (MccE492), an 84-amino acid antimicrobial peptide from Klebsiella pneumoniae has been purified in a post-translationally modified form from E. coli VCS257 harboring the pJAM229 plasmid or K. pneumoniae RCY492. The 831 Da modification was characterized by MS and NMR and found to consist of a trimer of N-(2,3-dihydroxybenzoyl)-L-serine linked via a C-glycosidic bond to b-D-glucose that was O-linked to the MccE492 (Thomas et al., 2004b) . It has been proposed that tunicamycin, an inhibitor of D-HexNAc-1-Ptranslocases, exerts its action by coordinating the divalent metal cofactor at the active site. MALDI spectra of tunicamycin in the presence of several divalent metals contained ions with compositions of [Tun þ Metal 2 ] þ showing that one of the metal atoms was present as a metal chelate (Xu et al., 2004) . The Cterminus loop 13 of the Na þ glucose co-transporter (SGLT1) has been shown to contain a binding site for inhibitory alkyl glucosides following photoaffinity labeling and MALDI-TOF analysis (Raja, Kipp, & Kinne, 2004) . Three hydroxyproline-rich peptides of 15, 18, and 20 amino acids, containing several pentose residues have been identified as defense signaling peptides released at wound sites in tomato plants (Pearce & Ryan, 2003) .

From the above, it can be seen that MALDI and particularly MALDI-TOF is still a major analytical method for carbohydrate analysis. Even though electrospray ionization, particularly when coupled with CID, is being increasingly used, MALDI-TOF usually provides a superior profile of constituent sugars because of the tendency of the technique to produce only singly charged ions. However, MALDI-TOF MS of native acidic glycans is less satisfactory because of problems with prompt fragmentation. Electrospray causes less fragmentation of these compounds but tends to produce ions in different charge states from glycans with several acidic groups, thus giving a profile that is not representative of the glycan content. However, this problem, and the instability of acidic carbohydrates under MALDI conditions, can be overcome by derivatization of the carboxylic acid group of sialic acids but sulfates must be protected by other techniques such as ion pairing. Thus, both MALDI and electrospray have advantages and disadvantages for carbohydrate work and the best technique to use will be dictated by the problem to be solved. The work during the review period has shown that although MALDI is now relatively ''mature,'' it is expected that future editions of this review series will be able to report many improved methods for performing these analyses. He is currently a consultant to the Department of Biochemistry, Oxford, the Oxford-Dublin Glycobiology Institute and is an Honorary Professorial Fellow at the University of Warwick, UK. He has served on the committee of the British Mass Spectrometry Society and has published more than 350 papers and reviews, mainly on mass spectrometry.

Enzymatic synthesis of complex glycosaminotrioses and study of their molecular recognition by hevein domains

Oligosaccharide and glycoprotein microarrays as tools in HIV glycobiology: Glycan-dependent gp120/protein interactions

Thioglycosylated cationic porphyrins-Convenient synthesis and photodynamic activity in vitro

A method for detecting Oglycanase in biological samples using a combination of MALDI-TOF mass spectrometry and time-resolved fluorimetry

High efficiency of transferring a native sugar chain from a glycopeptide by a microbial endoglycosidase in organic solvents

Ionone, iridoid and phenylethanoid glycosides from Ajuga salicifolia

Identification of N-glycosylation sites of the murine neural cell adhesion molecule NCAM by MALDI-TOF and MALDI-FTICR mass spectrometry

Yeast glycogenin (Glg2p) produced in Escherichia coli is simultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial glycogen accumulation

PimF, a mannosyltransferase of mycobacteria, is involved in the biosynthesis of phosphatidylinositol mannosides and lipoarabinomannan

Synthesis of tetra-and pentasaccharides corresponding to the capsular polysaccharide of Streptococcus pneumoniae type 9A&L, 9N and 9A

Synthesis of Cryptococcus neoformans capsular polysaccharide structures. IV. Construction of thioglycoside donor blocks and their subsequent assembly

Synthesis of neoglycoproteins containing O-methylated trisaccharides related to excretory/secretory antigens of Toxocara larvae

Improved capillary electrophoretic separation and mass spectrometric detection of oligosaccharides

Determination of Nglycosylation sites and site heterogeneity in glycoproteins

Synthesis of ganglioside epitopes for oligosaccharide specific immunoadsorption therapy of Guillian-Barré syndrome

Structural studies of the extracellular polysaccharide produced by Butyrivibrio fibrisolvens strain H10b

Determination of modulation of ligand properties of synthetic complex-type biantennary N-glycans by introduction of bisecting GlcNAc in silico, in vitro and in vivo

Default biosynthesis pathway for blood group-related glycolipids in human small intestine as defined by structural identification of linear and branched glycosylceramides in a group O Le(a-b-) nonsecretor

Detailed structural analysis of the peptidoglycan of the human pathogen Neisseria meningitidis

Study of the attachment of Na þ on glucose and on some of its methylated derivatives

Mycalosides B-I, eight new spermostatic steroid oligoglycosides from the sponge Mycale laxissima

Classification into a novel mollu-series of neutral glycosphingolipids from the lamp shell, Lingula unguis

Structural elucidation of novel phosphocholine-containing glycosylinositol-phosphoceramides in filamentous fungi and their induction of cell death of cultured rice cells

Newly discovered neutral glycosphingolipids in aureobasidin A-resistant zygomycetes: Identification of a novel family of GALA-series glycolipids with core Gal-1-6-Gal-1-6-Gal sequences

Synthesis of a branched chain aza-Cdisaccharide via the cycloaddition of a chiral nitrone to an alkene, both sugar derivatives

The glycosylation of human serum IgD and IgE and the accessibility of identified oligomannose structures for interaction with mannan binding lectin

Synthesis and cholera toxin binding properties of multivalent GM1 mimics

Preliminary investigation of the simultaneous detection of sugars, ascorbic acid, citric acid, and sodium benzoate in non-alcoholic beverages by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

An efficient approach towards the convergent synthesis of fully-carbohydrate mannodendrimers

Synthesis of sphere-type monodispersed oligosaccharide-polypeptide dendrimers

Synthesis of spherical and hemispherical sugar-containing poly(ornithine) dendrimers

Synthesis of structurally-controlled AIDS vaccine model with glyco-peptide dendrimer scaffolds

Porphyrin-containing glycodendrimers

Role of carbohydrate in stabilizing the triple-helix in a model for a deep-sea hydrothermal vent worm collagen

Characterization of the O-4-phosphorylated and O-5 substituted Kdo reducing end group and sequencing of the core oligosaccharide of Aeromonas salmonicida ssp salmonicida lipooligosaccharide using tandem mass spectrometry

N-Terminal segment of potato virus X coat protein subunits is glycosylated and mediates formation of a bound water shell on the virion surface

Immunoreactivity in mammals of two typical plant glyco-epitopes, core (1,3)-fucose and core xylose

Monoclonal C5-1 antibody produced in transgenic alfalfa plants exhibits a N-glycosylation that is homogenous and suitable for glycoengineering into human-compatible structures

Identification of the N-glycosylation sites on glutamate carboxypeptidase II necessary for proteolytic activity

2-Nitro thioglycoside donors: Versatile precursors of b-D-glycosides of aminosugars

Martix-assisted laser desorption time-of-flight mass spectrometry of dextran and dextrin derivatives

Matrix-assisted laser desorption/ ionization mass spectrometry with re-engineered 2,5-dihydroxybenzoic acid derivative

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. A comparison of fragmentation patterns of linear dextran obtained by in-source decay, post-source decay and collision-induced dissociation and the stability of linear and cyclic glucans studied by insource decay

Lipids of the zygomycete Absidia corymbifera F-965

Combinatorial carbohydrate synthesis

Expression of eukaryotic glycosyltransferases in the yeast Pichia pastoris

Specificity of IgG and IgE antibodies against plant and insect glycoprotein glycans determined with artificial glycoforms of human transferrin

A newly discovered cholesteryl galactoside from Borrelia burgdorferi

Chronic diabetes increases advanced glycation end products on cardiac ryanodine receptors/calcium-release channels

Controlled g-ray irradiation of heparin generates oligosaccharides enriched in highly sulfated sequences

Sialoside specificity of the siglec family assessed using novel multivalent probes. Identification of potent inhibitors of myelin-associated glycoprotein

Use of 15 N-NMR to resolve molecular details in isotopically-enriched carbohydrates: Sequence-specific observations in hyaluronan oligomers up to decasaccharides

Dendrimers in drug research

Engineering of an artificial glycosylation pathway blocked in core oligosaccharide assembly in the yeast Pichia pastoris: Production of complex humanized glycoproteins with terminal galactose

Human and bovine milk gangliosides differ in their fatty acid composition

Prebiotic concept for infant nutrition

Differential recognition of animal type b4-galactosylated and 3-fucosylated chito-oligosaccharides by two family 18 chitinases from Trichoderma harzianum

Mode of action of Fusarium moniliforme endopolygalacturonase towards acetylated pectin

Oglycosylation of a recombinant carbohydrate-binding module mutant secreted by Pichia pastoris

Replacement of carbohydrate sulfates by sugar C-sulfonic acid derivatives

Ion exchange and purification of carbohydrates on a Nafion(R) membrane as a new sample pretreatment for matrix-assisted laser desorption-ionization mass spectrometry

Differential effects of subunit asparagine 56 oligosaccharide structure on equine lutropin and follitropin hybrid conformation and receptor-binding activity

Glycerol and glycerol glycol glycodendrimers

Sugaring' carbosilane dendrimers via hydrosilylation

Genetic engineering of Pichia pastoris to humanize Nglycosylation of proteins

Matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry of oligosaccharides derivatized by reductive amination and N,N-dimethylation

Synthetic glycopeptides of the tandem repeat sequence of the epithelial mucin MUC4 with tumour-associated carbohydrate antigens

Synthetic tumor-associated glycopeptide antigens from the tandem repeat sequence of the epithelial mucin MUC4

Glycoforms of blactoglobulin with improved thermostability and preserved structural packing

Aminoglycoside array for the high-throughput analysis of small molecule-RNA interactions

Screening of sugar converting enzymes using quantitative MALDI-ToF mass spectrometry

Quantitative matrix-assisted laser desorption/ionization mass spectrometry for the determination of enzyme activities

The immunogenicity of the tumorassociated antigen Lewis y may be suppressed by a bifunctional crosslinker required for coupling to a carrier protein

Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis

Determination of carbohydrates in juices by capillary electrophoresis, high-performance liquid chromatography, and matrix-assisted laser desorption/ionizationtime of flight-mass spectrometry

Anionic adducts of oligosaccharides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Strategies for shotgun identification of posttranslational modifications by mass spectrometry

Methods for the analysis of hyaluronal and its fragments

In acute inflammation, the chondroitin-4 sulphate carried by bikunin is not only longer

Structural study of GCDFP/gp17 in disease versus physiological conditions using a proteomic approach

Structure of bacterial lipopolysaccharides

Identification and characterization of a novel cassava (Manihot esculenta Crantz) clone with high free sugar content and novel starch

Convenient methods for the synthesis of ferrocenecarbohydrate conjugates

Exopolysaccharides produced by a clinical strain of Burkholderia cepacia isolated from a cystic fibrosis patient

Analysis of a bioactive b-(1-3) polysaccharide (Curdlan) using matrix-assisted laser desorption/ionization time-offlight mass spectrometry

A straightforward synthesis of 1-adamantylmethyl glycosides, and their binding to cyclodextrins

Fructans in crested wheatgrass leaves

Molecularly imprinted TiO 2 -matrix-assisted laser desorption/ionization mass spectrometry for selectively detecting a-cyclodextrin

An efficient and practical synthesis of a-(1-3)-linked mannohexaose and mannooctaose

Differently sized granules from acetylated potato and sweet potato starches differ in the acetyl substitution pattern of their amylose populations

Characterization of a novel human UDP-GalNAc transferase, pp-GalNAc-T15

Characterizing biological products and assessing comparability following manufacturing changes

Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris

Nglycan structures of human transferrin produced by Lymantria dispar (gypsy moth) cells using the LdMNPV expression system

Characterisation of Mesorhizobium huakuii cyclic beta-glucan

Characterization of Mesorhizobium huakuii lipid A containing both D-galacturonic acid and phosphate residues

Synthesis of a cellobiosylated dimer and trimer and of cellobiose-coated polyamidoamine (PAMAM) dendrimers to study accessibility of an enzyme, cellodextrin phosphorylase

Identification of O-linked N-acetylglucosamine proteins in rat skeletal muscle using two-dimensional gel electrophoresis and mass spectrometry

The structure of the oligosaccharides of N-cadherin from human melanoma cell lines

Biosynthesis in vitro of Caenorhabditis elegans phosphorylcholine oligosaccharides

srf-3, a mutant of Caenorhabditis elegans, resistant to bacterial infection and to biofilm binding, is deficient in glycoconjugates

Synthesis of oligogalacturonates conjugated to BSA

Different behavior of dextrans in positive-ion and negative-ion mass spectrometry

Identity and localization of advanced glycation end products on human b 2 -microglobulin using matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry

Amino acid domains control the circulatory residence time of primate acetylcholinesterases in rhesus macaques (Macaca mulatta)

Comparative study of carbohydrate chains released from the oviducal mucins of the two closely related amphibian species Bombina bombina and Bombina variegata

Structure of lipid A from Pseudomonas corrugata by electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Glycosphingolipids in Plasmodium falciparum. Presence of an active glucosylceramide synthase

Development on new methodologies for the mass spectrometry study of bioorganic macromolecules

Cell transfection with polycationic cyclodextrin vectors

Synthesis of an arabinogalactan-type octa-and two isomeric nonasaccharides. Suitable tuning of protecting groups

Paradoxical impact of antioxidants on post-Amadori glycoxidation. Counterintuitive increase in the yields of pentosidine and N e -carboxymethyllysine using a novel multifunctional pyridoxamine derivative

Evaluation of newly synthesized and commercially available charged cyclomaltooligosaccharides (cyclodextrins) for capillary electrokinetic chromatography

Characterization of glycopeptides from HIV-I SF2 gp120 by liquid chromatography mass spectrometry

Localization of the O-glycosylated sites in peptides by fixed-charge derivatization with a phosphonium group

Matrixassisted laser desorption/ionization time-of-flight mass spectrometry and nuclear magnetic resonance analyses of end-functionalized saccharidic polymers: An example of a useful analytical technique combination

Molecular properties of hemicelluloses located in the surface and inner layers of hardwood and softwood pulps

Synthesis and characterisation of novel chromogenic substrates for human pancreatic a-amylase

Porphyromonas gingivalis lipopolysaccharide contains multiple lipid A species that functionally interact with both toll-like receptors 2 and 4

Polysaccharides of Pseudomonas pathovar strains that infect pea, tomato, and soya bean

Functional analysis of the ALG3 gene encoding the Dol-P-Man: Man 5 GlcNAc 2 -PP-Dol mannosyltransferase enzyme of P. pastoris

Structural determination of the O-chain moieties of the lipopolysaccharide fraction from Agrobacterium radiobacter DSM

A novel core region, lacking heptose and phosphate, of the lipopolysaccharide from the gram-negative bacterium Pseudomonas cichorii (Pseudomonadaceae RNA group 1)

Synthesis and conjugation of oligosaccharide analogues of fragments of the immunoreactive glycan part of the circulating anodic antigen of the parasite Schistosoma mansoni

Rapid chemoenzymatic synthesis of monodisperse hyaluronan oligosaccharides with immobilized enzyme reactors

Sulfated oligosaccharides isolated from the respiratory mucins of a secretor patient suffering from chronic bronchitis

Murine and human zona pellucida 3 derived from mouse eggs express identical O-glycans

Determination of glycopeptide structures by multistage mass spectrometry with low-energy collision-induced dissociation: Comparison of electrospray ionization quadrupole ion trap and matrix-assisted laser desorption/ionization quadrupole ion trap reflectron time-of-flight approaches

Synthesis of OSW-1 analogs with modified side chains and their antitumor activities

Monitoring glycosylation of therapeutic glycoproteins for consistency using highly fluorescent anthranilic acid

Revisiting the structure of the anti-neoplastic glucans of Mycobacterium bovis Bacille Calmette-Guérin: Structural analysis of the extracellular and boiling water extract-derived glucans of the vaccine substrains

Polysaccharides from grape berry cell walls. Part II. Structural characterization of the xyloglucan polysaccharides

Characterization of the carbohydrate moieties of the functional unit RvH 1 -a of Rapana venosa haemocyanin using HPLC/electrospray ionization MS and glycosidase digestion

Carbohydrate moieties of molluscan Rapana venosa hemocyanin

Structural and functional analysis of glycosylated Cu/Zn-superoxide dismutase from the fungal strain Humicola lutea 103

A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates

A new synthetic approach to mycobacterial cell wall a-(1 ! 5)-D-arabinofuranosyl C-oligosaccharides

A convenient synthesis of iminosugar-Cglycosides via organometallic addition to N-benzyl-N-glycosylhydroxylamines

Assembling heterocycle-tethered C-glycosyl and a-amino acid residues via 1,3-dipolar cycloaddition reactions

Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata

Synthesis of saponins using partially protected glycosyl donors

Deglycosylation of glycoproteins with trifluoromethanesulphonic acid: Elucidation of molecular structure and function

Phenolic glycoside isolated from seeds of the greater plantain (Plantago major L.)

Integrated selective enrichment target-A microtechnology platform for matrix-assisted laser desorption/ionization-mass spectrometry applied on protein biomarkers in prostate diseases

N-Acetylglucosamine-6-O-sulfotransferase-1: Production in the Baculovirus system and its applications to the synthesis of a sulfated oligosaccharide and to the modification of oligosaccharides in fibrinogen

Thalictricoside, a new phenolic compound from Thalictrum orientale

Physocalycoside, a new phenylethanoid glycoside from Phlomis physocalyx Hub.-Mor

Automated structural assignment of derivatized complex N-linked oligosaccharides from tandem mass spectra

Application of the StrOligo algorithm for the automated structure assignment of complex N-linked glycans from glycoproteins using tandem mass spectrometry

In vitro synthesis of a crystalline (1 ! 3,1 ! 4)-b-D-glucan by a mutated (1 ! 3,1 ! 4)-b-D-glucanase from Bacillus

A method for proteomic identification of membrane-bound proteins containing Asn-linked oligosaccharides

Regioselective synthesis of galactosylated tri-and tetrasaccharides by use of b-galactosidase from Bacillus circulans

Contribution of mass spectrometry to the study of the Maillard reaction in food

RuCl 3 -promoted amide formation from azides and thioacids

Assembly of sugars on polystyrene plates: A new facile microarray fabrication technique

Solid-state glycation of b-lactoglobulin by lactose and galactose: Localization of the modified amino acids using mass spectrometric techniques

Characterization of plant oligosaccharides by matrix-assisted laser desorption/ionization and electrospray mass spectrometry

Functional properties and application in peptide synthesis of trypsin modified with cyclodextrin-containing dicarboxylic acids

Biochemical and mass spectrometric characterization of soluble ecto-5 0 -nucleotidase from bull seminal plasma

Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells

The effects of ethanol on the glycosylation of human transferrin

N-and O-linked carbohydrates and glycosylation site occupancy in recombinant human granulocyte-macrophage colonystimulating factor secreted by a Chinese hamster ovary cell line

Irradiation effects in MALDI, ablation, ion production, and surface modifications. Part II: 2,5-dihydroxybenzoic acid monocrystals

Role of electrons in laser desorption/ionization mass spectrometry

Structural elucidation of zwitterionic carbohydrates derived from glycosphingolipids of the porcine parasitic nematode Ascaris suum

Overexpression of the waaZ Gene leads to modification of the structure of the inner core region of Escherichia coli lipopolysaccharide, truncation of the outer core, and reduction of the amount of O polysaccharide on the cell surface

Evaluation of sphingolipids in vitreous bodies from a patient with Gaucher disease, using delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry

N-linked glycan structures of human lactoferrin produced by transgenic rice

Chemoenzymatic synthesis of diverse asparagine-linked a-(2 ! 3)-sialyloligosaccharides

An efficient synthesis of a biantennary sialooligosaccharide analogue using a 1,6-anhydro-b-lactose derivative as a key synthetic block

Sensing behavior of fluorescent cyclodextrin/peptide hybrids bearing a macrocyclic metal complex

Separation and characterization of humic acids from Antarctia by capillary electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Inclusion complexes of humic acids with cyclodextrins

Chemoenzymatic synthesis of conformationally constrained oligosaccharides

Rapid screening of plasma volume expanders in urine using matrix-assisted laser desorption/ionisation time-offlight mass spectrometry

Enzymatic synthesis of the core-2 sialyl Lewis X O-glycan on the tumor-associated MUC1a' peptide

Analyzing response variability using high-pH anion-exchange chromatography and pulsed amperometric detection

Progresses of derivatization techniques for analyses of carbohydrates

Efficient preparation of glycoclusters from silsesquioxanes

Characterization of low-glycosylated forms of soluble human urokinase receptor expressed in Drosophila Schneider 2 cells after deletion of glycosylation-sites

Functional microdomains of glycolipids with partially fluorinated membrane anchors: Impact on cell adhesion

Stereoselective entry to b-linked C-disaccharides using a carbon-Ferrier reaction

Solutionand bound-state conformational study of N,N,N-triacetyl chitotriose and other analogous potential inhibitors of hevamine: Application of trNOESY and STD NMR spectroscopy

Advances in the production of human therapeutic proteins in yeasts and filamentous fungi

Glycosylation of human recombinant gonadotrophins: Characterization and batch-tobatch consistency

Characterization of keyhole limpet hemocyanin (KLH) glycans sharing a carbohydrate epitope with Schistosoma mansoni glycoconjugates

Cell wall polysaccharides of Brassica campestris seed cake: Isolation and structural features

Identification of a novel mannose-capped lipoarabinomannan from Amycolatopsis sulphurea

Tsukamurella paurometabola lipoglycan, a new lipoarabinomannan variant with pro-inflammatory activity

Acylation state of the phosphatidylinositol hexamannosides from Mycobacterium bovis bacillus Calmette Guérin and Mycobacterium tuberculosis H37Rv and its implication in toll-like receptor response

Are proton transfer reactions of excited states involved in UV laser desorption ionization?

Primary structure of the 2-Omethyl-a-L-fucose-containing side chain of the pectic polysaccharide, rhamnogalacturonan II

Differences among the cell wall galactomannans from Aspergillus wentii and Chaetosartorya chrysella and that of Aspergillus fumigatus

Metabolic incorporation of unnatural sialic acids into Haemophilus ducreyi lipooligosaccharides

Rapid oligosaccharide synthesis on a fluorous support

Molecular cloning and characterization of b1 ! 4-N-acetylgalactosaminyltransferases IV synthesizing N,N 0 -diacetyl-lactosediamine

Glycosylation and specific deamidation of ribonuclease B affect the formation of three-dimensional domain-swapped oligomers

AtBXL1, a novel higher plant (Arabidopsis thaliana) putative beta-xylosidase gene, is involved in secondary cell wall metabolism and plant development

Unique in vivo modifications of coagulation factor V produce a physically and functionally distinct platelet-derived cofactor

Efficient preparation of carbohydrate-and related polyol-amphiphiles by hydrazone ligation

Comparison of MALDI-TOF mass spectrometric to enzyme colorimetric quantification of glucose from enzyme-hydrolyzed starch

Nontypeable Haemophilus influenzae strain 2019 produces a biofilm containing N-acetylneuraminic acid that may mimic sialylated O-linked glycans

The glycosylated androgenic hormone of the terrestrial isopod Porcellio scaber (Crustacea)

Specific xyloglucanases as a new class of polysaccharidedegrading enzymes

Diffusion ordered spectroscopy as a complement to size exclusion chromatography in oligosaccharide analysis

Synthesis of 2-chloro-4-nitrophenyl a-Lfucopyranoside: A substrate for a-L-fucosidase (AFU)

Structural characterization of the lipid A component of Sinorhizobium sp. NGR234 rough and smooth form lipopolysaccharide. Demonstration that the distal amide-linked acyloxyacyl residue containing the long chain fatty acids is conserved in Rhizobium and Sinorhizobium sp

Molecular fingerprinting of carbohydrate structure phenotypes of three Porifera proteoglycan-like glyconectins

Lipomannan and lipoarabinomannan from a clinical isolate of Mycobacterium kansasii: Novel structural features and apoptosis-inducing properties

Solidsupported enzymatic synthesis of pectic oligogalacturonides and their analysis by MALDI-TOF mass spectrometry

Neutral N-glycans of the gastropod Arion lusitanicus

Glycopeptides as oligosaccharide mimics: High affinity sialopeptide ligands for sialoadhesin from combinatorial libraries

Structural characterization of TSC-36/ Flik. Analysis of two charge isoforms

Production of complex human glycoproteins in yeast

Polymer-resin hybrid capturerelease strategy for rapid oligosaccharide construction

Chemo-enzymatic synthesis and structureactivity study of artificially N-glycosylated eel calcitonin derivatives with a complex type oligosaccharide

Purification and characterization of mouse soluble receptor for advanced glycation end products (sRAGE)

O-Linked glycans control glycoprotein processing by antigenpresenting cells: A biochemical approach to the molecular aspects of MUC1 processing by dendritic cells

Synthesis and characterization of persilylated cyclodextrins

A novel synthesis of deoxy phospha sugar-sugar disaccharides

Quantitative aspects of the matrix-assisted laser desorption mass spectrometry of complex oligosaccharides

Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates

Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates and glycoconjugates

Analysis of carbohydrates and glycoconjugates by matrixassisted laser desorption/ionization mass spectrometry: An update covering the period 1999-2000

Analysis of carbohydrates and glycoconjugates by matrixassisted laser desorption/ionization mass spectrometry: An update covering the period

Fragmentation of Nlinked glycans with a MALDI-ion trap time-of-flight mass spectrometer

Conversion of pyridylamino sugar chains to 1-amino-1-deoxy derivatives, intermediates for tagging with fluorescein and biotin

Schizophyllans carrying oligosaccharide appendages as potential candidates for cell-targeted antisense carrier

Structural analysis of the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides

Structural characterization of the carbohydrate backbone of the lipopolysaccharide of Vibrio parahaemolyticus O-untypeable strain KX-V212 isolated from a patient

Structural elucidation of polysaccharide part of glycoconjugate from Treponema medium ATCC 700293

Chemical structure and immunobiological activity of lipid A from Prevotella intermedia ATCC 25611 lipopolysaccharide

Hallmarks of Caenorhabditis elegans N-glycosylation: Complexity and controversy

Synthesis of laminarin oligosaccharide derivatives having D-arabinofuranosyl side-chains

Endotoxic activity and chemical structure of lipopolysaccharides from Chlamydia trachomatis serotypes E and L 2 and Chlamydophila psittaci 6BC

Synthesis and characterisation of terminal carbohydrate modified poly(dimethylsiloxane)s

Specificity of Amaranthus leucocarpus syn. hypocondriacus lectin for O-glycopeptides

Altered glycosylation of a 1 -acid glycoprotein in patients with inflammation and diabetes mellitus

Targeted proteoglycomics analysis of sialyl Lewis X antigen expressing glycoproteins secreted by human hepatoma cell line

IgA nephropathy and tonsils-An approach from the structure of IgA1 produced by tonsillar lymphocytes

Affinity capturing and gene assignment of soluble glycoproteins produced by the nematode Caenorhabditis elegans

Preparation of a growth-promoting substance, lepidimoic acid, from okra pectic polysaccharide

A convenient synthesis of lepidimoide from okra mucilage and its growth-promoting activity in hypocotyls

A novel human b1 ! 3-Nacetylgalactosaminyltransferase that synthesizes a unique carbohydrate structure, GalNAcb1 ! 3GlcNAc

High-mannose-type oligosaccharides from human placental arylsulfatase A are core fucosylated as confirmed by MALDI MS

The first synthesis of peptide thioester carrying Nlinked core pentasaccharide through modified Fmoc thioester preparation: Synthesis of an N-glycosylated Ig domain of emmprin

Proteomic analysis of kappacasein micro-heterogeneity

Two glycosylation alterations of mouse intestinal mucins due to infection by the parasite Nippostrongylus brasiliensis

The Lec23 Chinese hamster ovary mutant is a sensitive host for detecting mutations in a-glucosidase I that give rise to congenital disorder of glycosylation IIb

IgA1 molecules produced by tonsillar lymphocytes are under-O-glycosylated in IgA nephropathy

The structural alteration of O-glycosylated hinge peptides in serum IgA1 before and after tonsillectomy in IgA nephropathy

Peptide and amino acid glycation: New insights into the Maillard reaction

An engineered hyaluronan synthase. Characterization of recombinant human hyaluronan synthase 2 expressed in Escherichia coli

Fluorescence resonance energy transfer in a novel cyclodextrin-peptide conjugate for detecting steroid molecules

Synthesis and immunochemical characterization of protein conjugates of carbohydrate and carbohydrate-mimetic peptides as experimental vaccines

Synthesis and biological activities of octyl 2,3-di-O-sulfo-a-L-fucopyranosyl-(1 ! 3)-2-O-sulfo-a-Lfucopyranosyl-(1 ! 4)-2,3-di-O-sulfo-a-L-fucopyranosyl-(1 ! 3)-2-Osulfo-a-L-fucopyranosyl-(1 ! 4)-2,3-di-O-sulfo-b-L-fucopyranoside

Synthesis and biological activities of octyl 2,3,4-tri-O-sulfo-a-L-fucopyranosyl-(1 ! 3)-2,4-di-O-sulfo-a-L-fucopyranosyl-(1 ! 3)-2,4-di-O-sulfo-a-L-fucopyranosyl-(1 ! 3)-2,4-di-O-sulfo-b-L-fucopyranoside

Application of MALDI-QTOF in proteomics and glycomics

Microscale nonreductive release of O-linked glycans for subsequent analysis through MALDI mass spectrometry and capillary electrophoresis

Ardisimamillosides G and H, two new triterpenoid saponins from Ardisia mamillata

Effects of polymerization initiator complexation in methacrylated b-cyclodextrin formulations

Analysis of site-specific glycosylation in recombinant human follistatin expressed in Chinese hamster ovary cells

Glycosylation of Rapana thomasiana hemocyanin. Comparison with other prosobranch (gastropod) hemocyanins

Efficient transport of saccharides through a liquid membrane mediated by a cyclodextrin dimer

Preparation and characterization of novel branched b-cyclodextrins having b-D-galactose residues on the non-reducing terminal of the side chains and their specific interactions with peanut (Arachis hypogaea) agglutinin

Molecular cloning and characterization of human GnT-IX, a Novel b1,6-N-acetylglucosaminyltransferase that is specifically expressed in the brain

N-Acetylglucosaminyltransferase IX acts on the GlcNAcb1,2-Mana1-Ser/Thr moiety, forming a 2,6-branched structure in brain O-mannosyl glycan

Synthesis of fullerene glycoconjugates through sulfide connection in aqueous media

Glycosphingolipids in edible fungi and their biological activities

Microwave-mediated analysis for sugar, fatty acid, and sphingoid compositions of glycosphingolipids

Recombinant (2 ! 3)-asialyltransferase immobilized on nickel-Agarose for preparative synthesis of sialyl Lewis x and Lewis a precursor oligosaccharides

Coupling thin-layer chromatography with vibrational cooling matrix-assisted laser desorption/ionization Fourier transform mass spectrometry for the analysis of ganglioside mixtures

Initiation of Oglycan synthesis in IgA1 hinge region is determined by a single enzyme, UDP-N-acetyl-a-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2

Synthesis of a hyaluronan neoglycopolymer by ring-opening metathesis polymerization

The Klebsiella pneumoniae wabG gene: Role in biosynthesis of the core lipopolysaccharide and virulence

Mannose-BSA conjugates: Comparison between commercially available linkers in reactivity and bioactivity

Isolation and characterization of water-soluble hemicelluloses from flax shive

Identification of post-translational modifications resulting from LHb polymorphisms by matrix-assisted laser desorption time-of-flight mass spectrometric analysis of pituitary LHb core fragment

Mass spectrometric characterization of a new 2-hydroxypropylb-cyclodextrin derivative bearing methacrylic moieties and its copolymerization with 1-vinyl-2-pyrrolidone

One-pot chemo-, regio-, and stereoselective double-differential glycosidation mediated by lanthanide triflates

Synthesis of a key Mycobacterium tuberculosis biosynthetic phosphoinositide intermediate

Carbohydrate biosensors

Modification-specific proteomics: Characterization of post-translational modifications by mass spectrometry

Specificity of enzymatic in vitro glycosylation by PNGase F: A comparison of enzymatic and nonenzymatic glycosylation

Structure of the xyloglucan produced by suspension-cultured tomato cells

Chemical characterization of different sugar-casein Maillard reaction products and protective effects on chemicalinduced cytotoxicity of Caco-2 cells

Glycosyltransferase assays utilizing N-acetyllactosamine acceptor immobilized on a cellulose membrane

Effect of silkworm hemolymph on N-linked glycosylation in two Trichoplusia ni insect cell lines

Synthesis of b-D-Galp-(1 ! 4)-b-D-GlcpNAc-(1 ! 2)-a-D-Manp-(1 ! O)(CH 2 ) 7 CH 3 mimics to explore the substrate specificity of sialyltransferases and trans-sialidases

Location of O-acetyl substituents in xylo-oligosaccharides obtained from hydrothermally treated Eucalyptus wood

Inner core assembly and structure of the lipooligosaccharide of Neisseria meningitidis: Capacity of strain NMB to express all known immunotype epitopes

Prompt chemoenzymatic synthesis of diverse complex-type oligosaccharides and its application to the solid-phase synthesis of a glycopeptide with Asn-linked sialyl-undeca-and asialo-nonasaccharides

Analysis of glycoproteins and the oligosaccharides thereof by high-performance capillary electrophoresis-Significance in regulatory studies on biopharmaceutical products

Inclusion complexes formed by sodium dodecyl sulfate and cyclodextrin-based nanotubes

Steroid polyols from the far eastern starfish Henricia sanguinolenta and H. leviuscula leviuscula

Analysis of the site-specific N-glycosylation of b1,6 Nacetylglucosaminyltransferase V

Detection of oligosaccharides labeled with cyanine dyes using matrix-assisted laser desorption/ionization mass spectrometry

Profiling analysis of oligosaccharides in antibody pharmaceuticals by capillary electrophoresis

Polyphenolics in Rhizophora mangle L. leaves and their changes during leaf development and senescence

a-Amylases of medical and industrial importance

Inhibitory effects of tannin on human salivary a-amylase

A novel b(1 ! 6)-N-acetylglucosaminyltransferase V (GnT-VB)

Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is a-D-Gal-(1 ! 3)-a-D-GalNAc

Relaxed sugar donor selectivity of a Sinorhizobium meliloti ortholog of the Rhizobium leguminosarum mannosyl transferase LpcC: Role of the lipopolysaccharide core in symbiosis of Rhizobiaceae with plants

A mannosyl transferase required for lipopolysaccharide inner core assembly in Rhizobium leguminosarum. Purification, substrate specificity and expression in Salmonella waaC mutants

Expression cloning and biochemical characterization of a Rhizobium leguminosarum lipid A 1-phosphatase

Biclonal systemic AL-amyloidosis with one glycosylated and one nonglycosylated AL-protein

Acylated flavonoid and phenylethanoid glycosides from Marrubium velutinum

Fluorous-tagged compound: A viable scaffold to prime oligosaccharide synthesis by cellular enzymes

In vitro biosynthesis of galactans by membrane-bound galactosyltransferase from radish (Raphanus sativus L.) seedlings

Study on the structures of xyloglucans using specific enzymes

Structural variability of BM-40/SPARC/osteonectin glycosylation: Implications for collagen affinity

Natural antibiotic function of a human gastric mucin against Helicobacter pylori infection

Malonylated flavonol glycosides from the petals of Clitoria ternatea

Flavonoid composition related to petal color in different lines of Clitoria ternatea

Post-source decay matrix-assisted laser desorption/ionization mass spectrometric study of peracetylated isoflavone glycosides cationized by protonation and with various metal ions

Cationization of simple organic molecules by singly-charged Ag 3 þ cluster ions in matrix-assisted laser desorption/ionization mass spectrometry: Metal cluster-molecule interactions

Synthesis of novel, multivalent glycodendrimers as ligands for HIV-1 gp120

A structural analysis of heparin-like glycosaminoglycans using MALDI-TOF mass spectrometry

Structural characterization of the Nglycan moiety and site of glycosylation in vitellogenin from the decapod crustacean Cherax quadricarinatus

Conjugate addition of phenols to 2-nitrogalactal-Synthesis of O-(2-acetamido-2-deoxygalactosyl)tyrosine

A convenient route to Oglycosyl lactates via conjugate addition to 2-nitroglycals: Ring closure to novel pyrano

An inositol-containing sphingolipid from the red alga Gracilaria verrucosa

A facile synthesis of novel cyclodextrin derivatives incorporating one b-(1,4)-glucosidic bond and their unique inclusion ability

Amphiphilic cyclodextrins as novel monosaccharide transport carriers through a bulk liquid membrane

Proteomic characterization of novel serum amyloid P component variants from human plasma and urine

Structural determination of the N-glycans of a lepidopteran arylphorin reveals the presence of a monoglucosylated oligosaccharide in the storage protein

Glycosylation of onconase increases its conformational stability and toxicity for cancer cells

Relative quantification of N-(carboxymethyl)lysine, imidazolone A, and the Amadori product in glycated lysozyme by MALDI-TOF mass spectrometry

Analysis of protein glycation products by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Synthesis and conformational characterization of the epidermal growth factor-like domain of blood coagulation factor IX carrying xylosyl-glucose

Chitosanolysis by a pectinase isozyme of Aspergillus niger-A non-specific activity

In situ liquid-liquid extraction as a sample preparation method for matrix-assisted laser desorption/ionization MS analysis of polypeptide mixtures

Glyco-SAMs as glycocalyx mimetics: Synthesis of L-fucose-and D-mannose-terminated building blocks

Function and glycosylation of plant-derived antiviral monoclonal antibody

Functional evaluation of carbohydrate-centred glycoclusters by enzyme-linked lectin assay: Ligands for concanavalin A

Influence of lactation parameters on the Nglycosylation of recombinant human C1 inhibitor isolated from the milk of transgenic rabbits

N-and O-glycans of recombinant human C1 inhibitor expressed in the milk of transgenic rabbits

Isolation and characterization of periplasmic cyclic b-glucans of Azorhizobium caulinodans

Biosynthesis of pectic galactan by membrane-bound galactosyltransferase from soybean (Glycine max Merr.) seedlings

Structure of a highly phosphorylated lipopolysaccharide core in the DalgC mutants derived from Pseudomonas aeruginosa wild-type strains PAO1 (serogroup O5) and PAC1R (serogroup O3)

High affinity capture surface for matrixassisted laser desorption/ionisation compatible protein microarrays

Targeted knockouts of Physcomitrella lacking plant specific immunogenic N-glycans

Identification of the required acyltransferase step in the biosynthesis of the phosphatidylinositol mannosides of Mycobacterium species

Purification and cDNA cloning of UDP-GlcNAc:GlcNAcb1 ! 3Galb1 ! 4Glc(NAc)-R [GlcNAc ! Gal]b1,6N-acetylglucosaminyltransferase from rat small intestine: A major carrier of dIGnT activity in rat small intestine

Polylactosamine synthesis and branch formation of N-glycans in b1,4-galactosyltransferase-1-deficient mice

N-Glycosylation pattern of the zymogenic form of human matrix metalloproteinase-9

Liquid chromatographic and mass spectrometric analysis of human serum acid alpha-1-glycoprotein

Trichoderma reesei acetyl esterase catalyzes transesterification in water

Mass spectrometric characterization of proteins from the SARS virus. A preliminary report

Site-specific N-glycosylation analysis: Matrix-assisted laser desorption/ionization quadrupole-quadrupole time-of-flight tandem mass spectral signatures for recognition and identification of glycopeptides

An improved model for prediction of retention times of tryptic peptides in ion pair reversed-phase HPLC. Its application to protein peptide mapping by off-line HPLC-MALDI MS

Matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry of heteropolyflavan-3-ols and glucosylated heteropolyflavans in Sorghum

Quantification of hyaluronic acid fragments in pharmaceutical formulations using LC-ESI-MS

Synthesis of stable dolichylphosphomannose analogues

Structural studies on IgG oligosaccharides of patients with primary Sjögren's syndrome

Structural characterization of Nglycopeptides by matrix-dependent selective fragmentation of MALDI-TOF/TOF tandem mass spectrometry

Post-translational modifications on proteins: Facile and efficient procedure for the identification of O-glycosylation sites by MALDI-LIFT-TOF/TOF mass spectrometry

Sequencing of N-linked oligosaccharides directly from protein gels: In-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high performance liquid chromatography

Identification of glycosylation sites in the SU component of the avian sarcoma/leukosis virus envelope glycoprotein (subgroup A) by mass spectrometry

Synthesis of the trisaccharide repeating unit of the atypical O-antigen polysaccharide from Danish Helicobacter pylori strains employing the 2 0 -carboxybenzyl glycoside

Synthesis of imino sugar scaffolds for the generation of glycosidase inhibitor libraries

Synthesis of the Lewis b hexasaccharide and HSA-conjugates thereof

Preparation and chiral recognition of a novel chiral stationary phase for high-performance liquid chromatography, based on mono(6 A -N-allylamino-6 A -deoxy)-perfunctionalized bcyclodextrin and covalently bonded silica gel

Derivatization of carbohydrates for chromatographic, electrophoretic and mass spectrometric structure analysis

Dynamic glycosylation of the transcription factor CREB: A potential role in gene regulation

Characterization of lecithin:cholesterol acyltransferase expressed in a human lung cell line

Enzymatic digestion and mass spectrometry in the study of advanced glycation end products/peptides

The complexity of non-enzymatic glycation product sets of human globins

Expression, secretion, and glycosylation of the 45-and 47-kDa glycoprotein of Mycobacterium tuberculosis in Streptomyces lividans

Structural characterization of the O-chain polysaccharide isolated from Bordetella avium ATCC 5086: Variation on a theme

Labelling saccharides with phenylhydrazine for electrospray and matrix-assisted laser desorption-ionization mass spectrometry

Profiling of N-linked oligosaccharides using phenylhydrazine derivatization and mass spectrometry

A new synthetic route for the preparation of glycoprobes

Hazelnut (Corylus avellana) vicilin Cor a 11: Molecular characterization of a glycoprotein and its allergenic activity

Melanoma cell CD44 interaction with the 1(IV) 1263-1277 region from basement membrane collagen is modulated by ligand glycosylation

Combination of two matrices results in improved performance of MALDI MS for peptide mass mapping and protein analysis

A study of the stability of tri(glucosyloxyphenyl)chlorin, a sensitizer for photodynamic therapy, in human colon tumoural cells: A liquid chromatography and MALDI-TOF mass spectrometry analysis

Transposon mutagenesis of Trypanosoma brucei identifies glycosylation mutants resistant to concanavalin A

Enantioseparation using cyclosophoraoses as a novel chiral additive in capillary electrophoresis

Cyclosophoraose as a catalytic carbohydrate for methanolysis

Antidiabetic effects of chitosan oligosaccharides in neonatal streptozotocin-induced noninsulin-dependent diabetes mellitus in rats

Antibodies that recognize bisected complex N-glycans on cell surface glycoproteins can be made in mice lacking N-acetylglucosaninyltransferase III

A mutation causing a reduced level of expression of six 4-galactosyltransferase genes is the basis of the Lec19 CHO glycosylation mutant

Structural analysis of lipid A from Escherichia coli O157:H7:K-using thin-layer chromatography and ion-trap mass spectrometry

The PmrA-regulated pmrC gene mediates phosphoethanolamine modification of lipid A and polymyxin resistance in Salmonella enterica

Synthesis and characterization of carboxymethylated cyclosophoraose, and its inclusion complexation behavior

Current chemical tagging strategies for proteome analysis by mass spectrometry

A genetic and structural analysis of the N-glycosylation capabilities of rice and other monocotyledons

A novel type of highly negatively charged lipooligosaccharide from Pseudomonas stutzeri OX1 possessing two 4,6-O-(1-carboxy)-ethylidene residues in the outer core region

Structure of minor oligosaccharides from the lipopolysaccharide fraction from Pseudomonas stutzeri OX1

Identification of mdoD, an mdoG paralog which encodes a twin-arginine-dependent periplasmic protein that controls osmoregulated periplasmic glucan backbone structures

Steroidal polyols from Far-Eastern starfishes Henricia sanguinolenta and H. leviuscula leviuscula

A new steroidal glycoside phrygioside A and its aglycone from the starfish Hippasteria phrygiana

Discovery and characterization of sialic acid O-acetylation in group B Streptococcus

Syntheses of arabinogalactans consisting of b-(1 ! 6)-linked D-galactopyranosyl backbone and a-(1 ! 3)-linked L-arabinofuranosyl side chains

Biotechnological production of highly soluble daidzein glycosides using Thermotoga maritima maltosyltransferase

Application of glycodiversification: Expedient synthesis and antibacterial evaluation of a library of kanamycin B analogues

Starch from hull-less barley: IV. Morphological and structural changes in waxy, normal and high-amylose starch granules during heating

Comparison of high-performance liquid chromatography and fluorophore-assisted carbohydrate electrophoresis methods for analyzing peptidoglycan composition of Escherichia coli

Synthesis of cluster mannosides via a Ugi four-component reaction and their inhibition against the binding of yeast mannan to concanavalin A

Expression of functional human transferrin in stably transfected drosophila S2 cells

A chemoenzymatic approach to glycopeptide antibiotics

Macrolactamization of glycosylated peptide thioesters by the thioesterase domain of tyrocidine synthetase

A method for the generation of glycoprotein mimetics

Inclusion complexation behavior of dyestuff guest molecules by a bridged bis(cyclomaltoheptaose)[bis(b-cyclodextrin)] with a pyromellitic acid diamide tether

Optimization of diagonal chromatography for recognizing post-translational modifications

An optimized protocol for nano-LC-MALDI-TOF-MS coupling for the analysis of proteolytic digests of glycoproteins

The IL-10R2 binding hot spot on IL-22 is located on the Nterminal helix and is dependent on N-linked glycosylation

Sialylation and sulfation of the carbohydrate chains in respiratory mucins from a patient with cystic fibrosis

GLYCO-FRAGMENT: A web tool to support the interpretation of mass spectra of complex carbohydrates

GlycoFragment and Glyco-SearchMS: Web tools to support the interpretation of mass spectra of complex carbohydrates

Molecular structures of fructans from Agave tequilana Weber var. azul

Preparation, properties, and applications of npentenyl arabinofuranosyl donors

Structure of a major glycolipid from Thermus oshimai NTU-063

AknK is an L-2-deoxyfucosyltransferase in the biosynthesis of the anthracycline aclacinomycin A

Synthesis and structural study of two new heparin-like hexasaccharides

Complement regulation at the molecular level: The structure of decayaccelerating factor

Serological characterization of anti-endotoxin serum directed against the conjugate of oligosaccharide core of Escherichia coli type R4 with tetanus toxoid

Identification of a 3-deoxy-D-manno-octulosonic acid biosynthetic operon in Moraxella catarrhalis and analysis of a KdsA-deficient isogenic mutant

Characterization of galactoglucomannan extracted from spruce (Picea abies) by heat-fractionation at different conditions

Effect of local matrix crystal variations in matrix-assisted ionization techniques for mass spectrometry

Mass spectrometric identification of N-and O-glycosylation sites of full-length rat selenoprotein P and determination of selenide-sulfide and disulfide linkages in the shortest isoform

Neoglycoconjugates from synthetic tetra-and hexasaccharides that mimic the terminus of the O-PS of Vibrio cholerae O:1, serotype Inaba

Concise synthesis of b-GlcNAcp-(1 ! 6)-a-Manp-(1 ! 6)-Manp and its dimer, b-GlcNAcp-(1 ! 2)-a-Manp-(1 ! 6)-Manp

Facile synthesis of arabinomannose penta-and decasaccharide fragments of the lipoarabinomannan of the equine pathogen, Rhodococcus equi

Recent developments in the synthesis and discovery of oligosaccharides and glycoconjugates for the treatment of disease

Synthesis of cis-(1 ! 3)-glycosides of allyl 2-acetamido-4,6-O-benzylidene-2-deoxy-a-D-glucopyranoside

Indiscriminate glycosylation of procarboxypeptidase Y expressed in Pichia pastoris

Matrix-assisted laser desorption and electrospray ionization mass spectrometry of carminic acid isolated from cochineal

Enzyme glycation influences product yields during oligosaccharide synthesis by reverse hydrolysis

Synthesis of oligosaccharides on soluble high-molecular-weight branched polymers in combination with purification by nanofiltration

Cloning and functional expression of a novel GDP-6-deoxy-D-talose synthetase from Actinobacillus actinomycetemcomitans

Casein phosphoproteome: Identification of phosphoproteins by combined mass spectrometry and two-dimensional gel electrophoresis

5-Dihydroxybenzoic acid butylamine and other ionic liquid matrixes for enhanced MALDI-MS analysis of biomolecules

Rapid synthesis of oligosaccharides using an anomeric fluorous silyl protecting group

Synthesis of the Lewis a trisaccharide based on an anomeric silyl fluorous tag

The pmrF polymyxin-resistance operon of Yersinia pseudotuberculosis is upregulated by the PhoP-PhoQ two-component system but not by PmrA-PmrB, and is not required for virulence

Basic amino acids as modulators of an O-linked glycosylation signal of the herpes simplex virus type 1 glycoprotein gC: Functional roles in viral infectivity

Synthesis, conformational studies and mannosidase stability of a mimic of 1,2-mannobioside

Galactosylated 5-hydroxylysine mimetics for glycopeptide synthesis

Structural characterization of chemically and enzymatically derived standard oligosaccharides isolated from partially purified tamarind xyloglucan

Synthesis of maltooligosyl fructofuranosides catalyzed by immobilized cyclodextrin glucosyltransferase using starch as donor

Identification of disulfide bonds and site-specific glycosylation in chicken a 1 -acid glycoprotein by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Structural characterization of oligosaccharides using MALDI-TOF/TOF tandem mass spectrometry

Glycoforms obtained by expression in Pichia pastoris improve cancer targeting potential of a recombinant antibody-enzyme fusion protein

Expression, purification, and characterization of avian Thy-1 from Lec1 mammalian and Tn5 insect cells

Cellular effects of deoxynojirimycin analogues: Uptake, retention and inhibition of glycosphingolipid biosynthesis

Cellular effects of deoxynojirimycin analogues: Inhibition of Nlinked oligosaccharide processing and generation of free glucosylated oligosaccharides

A scavenger function for a Drosophila peptidoglycan recognition protein

Tailoring modification of deoxysugars during biosynthesis of the antitumour drug chromomycin A

Assembly of a library of trisaccharides as mimics of sialyl Lewis X via random combination strategy

Chemical and enzymatic release of glycans from glycoproteins

O-Glycan sialylation and the structure of the stalk-like region of the T cell co-receptor CD8

Specificities of three distinct human chondroitin/dermatan N-acetylgalactosamine 4-O-sulfotransferases demonstrated using partially desulfated dermatan sulfate as an acceptor: Implication of differential roles in dermitan sulfate biosynthesis

The underglycosylation of plasma 1-antitrypsin in congenital disorders of glycosylation type I is not random

Mass spectrometric analysis of glycans in elucidating the pathogenesis of CDG type IIx

5-Amino-2-mercapto-1,3,4-thiadiazole: A new matrix for the efficient matrixassisted laser desorption/ionization of neutral carbohydrates

Estimation of the proton affinity values of fifteen matrix-assisted laser desorption/ionization matrices under electrospray ionization conditions using the kinetic method

Nlinked glycan structures of mouse interferon-b produced by Bombyx mori larvae

Structural and functional characterization of ovotransferrin produced by Pichia pastoris

New polygalacturonases from Trichoderma reesei: Characterization and their specificities to partially methylated and acetylated pectins

A transient tobacco expression system coupled to MALDI-TOF-MS allows validation of the impact of differential targeting on structure and activity of a recombinant therapeutic glycoprotein produced in plants

The structure of lipid A of the lipopolysaccharide from Burkholderia caryophylli with a 4-amino-4-deoxy-L-arabinopyranose-1-phosphate residue exclusively in glycosidic linkage

Sample preparation effects in matrix-assisted laser desorption/ ionisation time-of-flight mass spectrometry of partially depolymerised carboxymethyl cellulose

Sample preparation effects in matrix-assisted laser desorption/ ionisation time-of-flight mass spectrometry of partially depolymerised methyl cellulose

Phase-variation of the truncated lipo-oligosaccharide of Neisseria meningitidis NMB phosphoglucomutase isogenic mutant NMB-R6

Structure of the major triterpene glycoside from the sea cucumber Stichopus mollis and evidence to reclassify this species into the new genus Australostichopus

Sequencing of oligosaccharides derivatized with benzylamine using electrospray ionization-quadrupole time of flight-tandem mass spectrometry

Fragmentation characteristics of permethylated oligosaccharides using a matrix-assisted laser desorption/ionization twostage time-of-flight (TOF/TOF) tandem mass spectrometer

Enzymatic preparation of biotinylated naturally-occurring sialylglycan and its molecular recognition on a quartz-crystal microbalance

Biosynthesis of mycobacterial phosphatidylinositol mannosides

PmrAB, a two-component regulatory system of Pseudomonas aeruginosa that modulates resistance to cationic antimicrobial peptides and addition of aminoarabinose to lipid A

Structural analysis of kappacarrageenan oligosaccharides released by carrageenase from marine cytophaga MCA-2

Atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) on a quadrupole ion trap mass spectrometer

Aggregates are the biologically active units of endotoxin

Synthesis of peptide dendrimers based on a b-cyclodextrin core with guest binding ability

Total synthesis of a tetra-and two pentasaccharide fragments of the O-specific polysaccharide of Shigella flexneri serotype 2a

Photocontrolled release and uptake of a porphyrin guest by dithienylethene-tethered -cyclodextrin host dimers

Effects of buffering conditions and culture pH on production rates and glycosylation of clinical phase I anti-melanoma mouse IgG3 monoclonal antibody R24

Enhanced post-source decay and cross-ring fragmentation of oligosaccharides facilitated by conversion to amino derivatives

Capillary electrophoresis to characterize ricin and its subunits with matrixassisted laser desorption/ionization time-of-flight mass spectrometry

Glucose production by hydrolysis of starch under hydrothermal conditions

Construction of artificial signal transducers on a lectin surface by post-photoaffinity-labeling modification for fluorescent saccharide biosensors

Synthesis of an unnatural N-glycan-linked dolichyl pyrophosphate precursor

Cyclodextrin trimers as receptors for arranging ester and catalyst at optimized location to achieve enhancement of hydrolytic activity

Synthesis of glycosyl glycerol by cyclodextrin glucanotransferases

Sample clean-up method for analysis of complex N-glycans released from glycopeptides

Detailed structural features of glycan chains derived from a1-acid glycoproteins of several different animals: The presence of hypersialylated, O-acetylated sialic acids but not disialyl residues

Structural analysis of N-linked glycans in Caenorhabditis elegans

Dynamic multivalent lactosides displayed on cyclodextrin beads dangling from polymer strings

Synthesis of lactoside glycodendrons using photoaddition and reductive amination methodologies

Flavonoids of Hedysarum setigerum

Hydrolysis of Nothogenia erinacea xylan by xylanases from families 10 and 11

Expression, purification and characterization of humanized anti-HBs Fab fragment

An efficient method for the synthesis of 2,6-branched galacto-oligosaccharides and its applications to the synthesis of three tetrasaccharides and a hexasaccharide related to the arabinogalactans (AGs)

Design and synthesis of C 3 -symmetric Lewis X antigen

Living ringopening metathesis polymerization of norbornenes containing acetylprotected carbohydrates using well-defined molybdenum and ruthenium initiators

Flavone C-glycoside, phenolic acid, and nitrogen contents in leaves of barley subject to organic fertilization treatments

In situ extension as an approach for identifying novel a-amylase inhibitors

Immunity to schistosomiasis: Glycans are potential antigenic targets for immune intervention

Solid-phase extraction NMR studies of chromatographic fractions of saponins from Quillaja saponaria

Enzymatic glycosidation of sugar oxazolines having a carboxylate group catalyzed by chitinase

Enzymatic synthesis of alternatingly 6-O-carboxymethylated chitotetraose by selective glycosidation with chitinase catalysis

Analysis of sugar epimers using mass spectrometry: N-acetyllactosamine-6-6 0 -disulfate and the 2 0 -epimer

N-Glycosylation patterns of HSA/CD24 from different cell lines and brain homogenates: A comparison

Glycotentacles: Synthesis of cyclic glycopeptides, toward a tailored blocker of influenza virus hemagglutinin

Carbohydrate structure and differential binding of prostate specific antigen to Maackia amurensis lectin between prostate cancer and benign prostate hypertrophy

Simple formylacetal (CH 2 ) as a novel linker for saccharide synthesis on soluble-polymer support

Synthesis of heparinlike oligosaccharides on polymer supports

Synthesis and structural analysis of five novel oligosaccharides prepared by glucosyltransfer from b-D-glucose 1-phosphate to isokestose and nystose using Thermoanaerobacter brockii kojibiose phosphorylase

Identification and removal of O-linked and non-covalently linked sugars from recombinant protein produced using Pichia pastoris

Structural evidence of grafting in electron beam irradiated starch-allylurea blends

Artefacts formed by addition of urea to N-linked glycans released with peptide-N-glycosidase F for analysis by mass spectrometry

Structural features of acetylated galactomannans from green Coffea arabica beans

Synthesis and biological activities in vitro and in vivo of glycosylated human interleukin-1a, neoglyco IL-1a, coupled with N-acetyloneuraminyl-galactose

Diverse motifs of mannoside clustering on a b-cyclodextrin core

Anion complexation by glycocluster thioureamethyl cavitands: Novel ESI-MS-based methods for the determination of K a values

The first representative of glycosylated three-fingered toxins. Cytotoxin from the Naja kaouthia cobra venom

Generation of new landomycins by combinatorial biosynthetic manipulation of the LndGT4 gene of the landomycin E cluster in S. globisporus

Mucor hiemalis endo-b-N-acetylglucosaminidase can transglycosylate a bisecting hybrid-type oligosaccharide from an ovalbumin glycopeptide

Design of attachment type of drug delivery system by complex formation of avidin with biotinyl drug model and biotinyl saccharide

Increasing sensitivity and decreasing spot size using an inexpensive, removable hydrophobic coating for matrix-assisted laser desorption/ionisation plates

Separation of hemicellulosic oligomers from steamtreated spruce wood using gel filtration

Automated synthesis of oligosaccharides

In vitro glycosylation of peptide (RKDVY) and RNase A by PNGase F

Identification of the binding site of methylglyoxal on glutathione peroxidase: Methylglyoxal inhibits glutathione peroxidase activity via binding to glutathione binding sites Arg 184 and 185

Characterization of a lysosomal serine carboxypeptidase from Trypanosoma cruzi

Molecular basis of anti-horseradish peroxidase staining in Caenorhabditis elegans

Novel bacterial polar lipids containing ether-linked alkyl chains, the structures and biological properties of the four major glycolipids from Propionibacterium propionicum PCM 2431 (ATCC 14157 T )

Structural and serological characterization of the major glycolipid from Rothia mucilaginosa

USO de espectroscopia de rmn y maldi-tof ms en la elucidacion estructural de flavonoides antioxidantes provenientes de la planta medicinal chilena Cheilanthes glauca

Mannose metabolism is required for mycobacterial growth

Reiterative intramolecular glycosylation supported by a rigid spacer

Systemic signaling in tomato plants for defense against herbivores: Isolation and characterization of three novel defense-signaling glycopeptide hormones coded in a single precursor gene

Synthesis of sucrose laurate using a new alkaline protease

New class of quaternary ammonium salts, derivatives of methyl D-glucopyranosides

Roles of 3-deoxy-D-manno-2-octulosonic acid transferase from Moraxella catarrhalis in lipooligosaccharide biosynthesis and virulence

Glycosylation of human pancreatic ribonuclease: Differences between normal and tumor states

Altered glycosylation pattern allows the distinction between prostate-specific antigen (PSA) from normal and tumor origins

Studies of new polysaccharides from Lasallia pustulata (L.) Hoffm

Molecular dissection of the role of two methyltransferases in the biosynthesis of phenolglycolipids and phthiocerol dimycoserosate in the Mycobacterium tuberculosis complex

Characterization of three glycosyltransferases involved in the biosynthesis of the phenolic glycolipid antigens from the Mycobacterium tuberculosis complex

Synthesis and conformational analysis of novel N(OCH 3 )-linked disaccharide analogues

Sample preparation for hyphenated analytical techniques

Minor interspecies variations in the sequence of the gp53 TSL-1 antigen of Trichinella define species-specific immunodominant epitopes

Two-dimensional electrophoresis replica blotting: A valuable technique for the immunological and biochemical characterization of single components of complex extracts

Purification and characterization of a b-xylosidase from potatoes (Solanum tuberosum)

Mass spectrometric assignment of Smith degradation glycopeptides derived from ribonuclease B

Stemmosides C and D, two novel unusual pregnane glycosides from Solenostemma argel: Structural elucidation and configurational study by a combined NMR-quantum mechanical strategy

N-glycosylated catalytic unit meets O-glycosylated propeptide: Complex protein architecture in a fungal hexosaminidase

Glycosylation profile of integrin a3b1 changes with melanoma progression

Chemical contributions to understanding heparin activity: Synthesis of related sulfated oligosaccharides

A novel mannitol teichoic acid with side phosphate groups of Brevibacterium sp. VKM Ac-2118

The high CO 2 -solubility of per-acetylated a-, b-and gcyclodextrin

Glycoform composition profiling of O-glycopeptides derived from human serum IgA1 by matrix-assisted laser desorption ionization-time of flight-mass spectrometry

New set of orthogonal protecting groups for the modular synthesis of heparan sulfate fragments

Mass spectrometric methods for the determination of flavonods in biological samples

Characterizing the electrospray-ionization mass spectral fragmentation pattern of enzymatically derived hyaluronic acid oligomers

Bordetella bronchiseptica PagP is a Bvg-regulated lipid A palmitoyl transferase that is required for persistent colonization of the mouse respiratory tract

Efficient synthesis of unsymmetrical ureido-linked disaccharides

Matrixassisted laser desorption/ionization-time of flight-mass spectrometry of lipopolysaccharide species separated by slab-polyacrylamide gel electrophoresis: High-resolution separation and molecular weight determination of lipooligosaccharides from Vibrio fischeri strain HMK

Characterization of a tomato protein that inhibits a xyloglucan-specific endoglucanase

An outer membrane enzyme that generates the 2-amino-2-deoxy-gluconate moiety of Rhizobium leguminosarum lipid A

A methylated phosphate group and four amide-linked acyl chains in Leptospira interrogans lipid A: The membrane anchor of an unusual lipopolysaccharide that activates TLR2

C-terminus loop 13 of Na þ glucose cotransporter SGLT1 contains a binding site for alkyl glucosides

The heparin/heparan sulfate 2-O-sulfatase from Flavobacterium heparinum. A structural and biochemical study of the enzyme active site and saccharide substrate specificity

New access to lipo-chitooligosaccharide nodulation factors

b-N-Acetylhexosaminidase-catalysed synthesis of non-reducing oligosaccharides

Enzymatic synthesis of N-acetylglucosaminobioses by reverse hydrolysis: Characterisation and application of the library of fungal b-N-acetylhexosaminidases

Structural investigation of hemicellulosic polysaccharides from Argania spinosa: Characterisation of a novel xyloglucan motif

A gene, uge, is essential for Klebsiella pneumoniae virulence

Synthesis of peptide and glycopeptide partial structures of the homophilic recognition domain of epithelial cadherin

Structural characterisation by MALDI-MS of olive xylo-oligosaccharides obtained by partial acid hydrolysis

Introducing transglycosylation activity into human salivary aamylase (HSA)

Enzymatic supported synthesis of lacto-N-neotetraose using dendrimeric polyethylene glycol

Lipase-catalysed regioselective acylations in combination with regioselective glycosylations as a strategy for the synthesis of oligosaccharides: Synthesis of a series of fucosyllactose building blocks

Characterisation of the substituent distribution in starch and cellulose derivatives

Phosphorylation of transitory starch is increased during degradation

A programmable one-pot oligosaccharide synthesis for diversifying the sugar domains of natural products: A case study of vancomycin

Microscale analysis of mucin-type O-glycans by a coordinated fluorophore-assisted carbohydrate electrophoresis and mass spectrometry approach

Evidence of regio-specific glycosylation in human intestinal mucins: Presence of an acidic gradient along the intestinal tract

Efficacy of enzyme replacement therapy in a-mannosidosis mice: A preclinical animal study

Donor specificity in the glycosylation of Tamm-Horsfall glycoprotein: Conservation of the Sd a determinant in pairs of twins

Chemically methylated and reduced pectins: Preparation, characterisation by 1 H NMR spectroscopy, enzymatic degradation, and gelling properties

Derivatization in the current practice of analytical chemistry

Cyclodextrins containing an acetone bridge. Synthesis and study as epoxidation catalysts

Cu(II)-Self-assembling bipyridyl-glycoclusters and dendrimers bearing the Tn-antogen cancer marker: Synthesis and lectin binding properties

Secretory IgA N-and O-glycans provide a link between the innate and adaptive immune systems

Two carbohydrate binding sites in the H cc -domain of tetanus neurotoxin are required for toxicity

Aminoglycoside-derived cationic lipids for gene transfection: Synthesis of kanamycin A derivatives

Sugars within a hydrophobic scaffold: Glycodendrimers from polyphenylenes

One-pot preparation of a series of glycoconjugates with predetermined antigen-carrier ratio from oligosaccharides that mimic the O-PS of Vibrio cholerae O:1, serotype Ogawa

A practical synthesis of amphiphilic cyclodextrins fully substituted with sugar residues on the primary face

EPR and affinity studies of mannose-TEMPO functionalized PAMAM dendrimers

Comparative analysis of the site-specific N-glycosylation of human lactoferrin produced in maize and tobacco plants

Separation of recombinant human erythropoietin glycoforms by capillary electrophoresis using volatile electrolytes. Assessment of mass spectrometry for the characterization of erythropoietin glycoforms

Synthesis of new peptidic glycoclusters derived from b-alanine

Differential roles of two N-acetylgalactosaminyltransferases, CSGalNAcT-1, and a novel enzyme, CSGalNAcT-2. Initiation and elongation in synthesis of chondroitin sulfate

Molecular cloning and characterization of a novel human b1,4-N-acetylgalactosaminyltransferase, b4GalNAc-T3, responsible for the synthesis of N,N 0 -diacetyllactosediamine, GalNAcb1 ! 4Glc-4GlcNAc

Glycoinsulins: Dendritic sialyloligosaccharide-displaying insulins showing a prolonged blood-sugar-lowering activity

Site-specific introduction of sialic acid into insulin

Display of azido glycoside on a sensor chip

N-glycosylation at Asn in the Asn-Xaa-Cys motif of human transferrin

Fast and efficient synthesis of a novel homologous series of L-fucosylated trisaccharides using the Helix pomatia a-(1 ! 2)-L-galactosyltransferase

Enzymatic a(1 ! 2)-L-fucosylation: Investigation of the specificity of the a(1 ! 2)-Lgalactosyltransferase from Helix pomatia

Facile synthesis of b(1 ! 6)-D-galactosylated oligosaccharides employing the b(1 ! 6)-D-GalT-II from the albumen gland of the snails Helix pomatia

Structure of pre-S2 N-and O-linked glycans in surface proteins from different genotypes of hepatitis B virus

Chiral capillary electrophoresis: Facts and fiction on the reproducibility of resolution with randomly substituted cyclodextrins

Acylated cholesteryl galactoside as a novel immunogenic motif in Borrelia burgdorferi Sensu Stricto

Synthesis of oligosaccharides as potential novel food components and upscaled enzymatic reaction employing the b-galactosidase from bovine testes

The obligate predatory Bdellovibrio bacteriovorus possesses a neutral lipid A containing a-Dmannoses that replace phosphate residues: Similarities and differences between the lipid As and the lipopolysaccharides of the wild type strain B. bacteriovorus HD100 and its host-independent derivative HI100

Post-translational modifications and their biological functions: Proteomic analysis and systematic approaches

Formation of phosphatidic acid, ceramide, and diglyceride on radiolysis of lipids: Identification by MALDI-TOF mass spectrometry

A complex array of post-translation modifications determines the circulatory longevity of acetylcholinesterase in a hierarchical manner

Chemical synthesis of a skeleton structure sperm CD52-A GPI-anchored glycopeptide

A mass spectrometry plate reader: Monitoring enzyme activity and inhibition with a desorption/ionization on silicon (DIOS) platform

The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity

Direct Nglycan profiling in the presence of tryptic peptides on MALDI-TOF by controlled ion enhancement and suppression upon glycan-selective derivatization

Neoculin as a new tastemodifying protein occurring in the fruit of Curculigo latifolia

Pseudostichoposide B-New triterpene glycoside with unprecedent type of sulfatation from the deep-water North Pacific sea cucumber Pseudostichopus trachus

Full structural characterization of the lipid A components from the Agrobacterium tumefaciens strain C58 lipopolysaccharide fraction

The complete structure of the lipooligosaccharide from the halophilic bacterium Pseudoalteromonas issachenkonii KMM 3549 T

The structure of the phosphorylated carbohydrate backbone of the lipopolysaccharide of the phytopathogen bacterium Pseudomonas tolaasii

The structures of the lipid A moieties from the lipopolysaccharides of two phytopathogenic bacteria, Xanthomonas campestris pv. pruni and Xanthomonas fragariae

Structure elucidation of the highly heterogeneous lipid A from the lipopolysaccharide of the gram-negative extremophile bacterium Halomonas magadiensis strain 21 M1

Structure of a heteroxylan of gum exudate of the palm Scheelea phalerata (uricuri)

How does peripheral lipopolysaccharide induce gene expression in the brain of rats

Attachment of glycosaminoglycan oligosaccharides to thiolderivatised gold surfaces

Synthesis and structure of selected quaternary N-(1,4-anhydro-5-deoxy-2,3-O-isopropylidene-D,L-ribitol-5-yl)ammonium salts

Characterization of extracellular polymers of Phaeocystis globosa and P. antarctica

Enantioselectivity in gas-phase ion-molecule reactions

New fragmentation mechanisms in matrixassisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry of carbohydrates

Recombinant anti-hCG antibodies retained in the endoplasmic reticulum of transformed plants lack core-xylose and corea(1,3)-fucose residues

Synthesis of the first fully active lipoteichoic acid

Matrix-assisted laser-desorption time-of flight ionization and high-performance liquid chromatography-electrospray ionization mass spectral analysis of two glycosylated recombinant epoetins

Fragmentation characteristics of neutral N-linked glycans using a MALDI-TOF/TOF tandem mass spectrometer

The N-linked oligosaccharides of aminopeptidase N from Manduca sexta. Site localization and identification of novel N-glycan structures

Determination of neutral oligosaccharides in vegetables by matrix-assisted laser desorption/ionization mass spectrometry

Human galectin-1 recognition of poly-N-acetyllactosamine and chimeric polysaccharides

Unaltered complex Nglycan profiles in Nicotiana benthamiana despite drastic reduction of b1,2-N-acetylglucosaminyltransferase I activity

Generation of Arabidopsis thaliana plants with complex N-glycans lacking b1,2-linked xylose and core a1,3-linked fucose

MALDI: More than peptide mass fingerprints

Structural analysis of arabinoxylans isolated from native and malted finger millet (Eleusine coracana, ragi)

Refinement of the structures of cell-wall glucans of Schizosaccharomyces pombe by chemical modification and NMR spectroscopy

vAL-1, a novel polysaccharide lyase encoded by chlorovirus CVK2

Synthesis of dihydrodiosgenin glycosides as mimetics of bidesmosidic steroidal saponins

Synthesis of rhamnosylated diosgenyl glucosides as mimetics of cytostatic steroidal saponins from Ornithogalum saundersiae and Galtonia candicans

Selective detection of glycopeptides on ion trap mass spectrometers

Site-specific N-glycosylation of chicken serum IgG

Preparation of a unique glucan with large intervals in molecular weight distribution. Controlled ring-opening polymerization of O-permethylcyclodextrin

N-Glycan structures of pigeon IgG. A. major serum glycoprotein containing Gala1 ! 4 termini

Capillary electrophoresis of carbohydrates

Enzymatic synthesis of lipid A molecules with four amidelinked acyl chains. LpxA acetyltransferases selective for an analog of UDP-N-acetylglucosamine in which an amine replaces the 3 00 -hydroxyl group

A novel method for the separation and purification of human serum acid alpha-1-glycoprotein. liquid chromatographic and mass spectrometric investigation of tryptic fragments

Structure of oligosaccharide side chains of an intestinal immune system modulating arabinogalactan isolated from rhizomes of Atractylodes lancea DC

Transient production of recombinant proteins by Chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents

Potato D-enzyme catalyzes the cyclization of amylose to produce cycloamylose, a novel cyclic glucan

Conversion of pyridylamino sugar chains to corresponding reducing sugar chains

N-Glycan structures of squid rhodopsin. Existence of the a1-3 and a1-6 difucosylated innermost GlcNAc residue in a molluscan glycoprotein

Abnormal biantennary sugar chains are expressed in human chorionic gonadotropin produced in the choriocarcinoma cell line

Solid-phase synthesis of core 2 O-linked glycopeptide and its enzymatic sialylation

Synthesis of a mimic for the heterogeneous surface of core 2 sialoglycan-linked glycoprotein

Synthesis of chondroitin sulfate E hexasaccharide in the repeating region by an effective elongation strategy toward longer chondroitin oligosaccharide

Mass spectrometry of N-linked oligosaccharides using atmospheric pressure infrared laser ionization from solution

Studies directed toward the synthesis of protein-bound GPI anchor

Human serum IgA1 is substituted with up to six O-glycans as shown by matrix assisted laser desorption ionisation time-of-flight mass spectrometry

Microdeposition device interfacing capillary electrochromatography and microcolumn liquid chromatography with matrix-assisted laser desorption/ionization mass spectrometry

Isolation and characterization of O-acetylated glucomannans from aspen and birch wood

Regioselective acylation of ginsenosides by Novozyme 435

Regioselective acylation of ginsenosides by Novozyme 435 to generate molecular diversity

A facile synthesis of a novel polyacetal containing trehalose residue in the main chain

Practical and convenient modifications of the A,Csecondary hydroxyl face of cyclodextrins

Cyclodextrin-bound 6-(4-methoxyphenyl) imidazo[1,2-a]pyrazin-3(7H)-ones with fluorescein as green chemiluminescent probes for superoxide anions

Mechanism of 2-O-3-O silyl migration in cyclomaltohexaose (a-cyclodextrin)

Formation of squaric acid amides of anthracycline antibiotics. Synthesis and cytotoxic properties

A new type of congenital disorders of glycosylation (CDG-Ii) provides new insights into the early steps of dolichol-linked oligosaccharide biosynthesis

Production of a complement inhibitor possessing sialyl Lewis X moieties by in vitro glycosylation technology

Siderophore peptide, a new type of post-translationally modified antibacterial peptide with potent activity

A catalogue of molecular, physiological and symbiotic properties of soybeannodulating rhizobial strains from different soybean cropping areas of China

Physicochemical characterization of the endotoxins from Coxiella burnetii strain Priscilla in relation to their bioactivities

Complex-type biantennary N-glycans of recombinant human transferrin from Trichoplusia ni insect cells expressing mammalian b-1,4-galactosyltransferase and b-1,2-Nacetylglucosaminyltransferase II

Comparing N-glycan processing in mammalian cell lines to native and engineered lepidopteran insect cell lines

Periplasmic cleavage and modification of the 1-phosphate group of Helicobacter pylori lipid A

Characterization of N-and O-linked glycosylation of recombinant human bile salt-stimulated lipase secreted by Pichia pastoris

Catalog-library approach for the rapid and sensitive structural elucidation of oligosaccharides

Synthesis of photoaffinity probes [2 0 -iodo-4 0 -(3 00 -trifluoromethyldiazirinyl)phenoxy]-D-glucopyranoside and [(4 0 -benzoyl)phenoxy]-D-glucopyranoside for the identification of sugarbinding and phlorizin-binding sites in the sodium/-glucose cotransporter protein

The MisR/MisS two-component regulatory system influences inner core structure and immunotype of lipooligosaccharide in Neisseria meningitidis

Determination of phosphorylation sites in lipooligosaccharides from Pseudoalteromonas haloplanktis TAC 125 grown at 158C and 258C by nano-electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Phaiodactylipin, a glycosylated heterodimeric phospholipase A 2 from the venom of the scorpion Anuroctonus phaiodactylus

Advanced glycation end product ligands for the receptor for advanced glycation end products: Biochemical characterization and formation kinetics

Partially esterified oligogalacturonides are the preferred substrates for pectin methylesterase of Aspergillus niger

Synthesis of novel trivalent amino acid glycoconjugates based on the cyclotriveratrylene ('CTV') scaffold

Substitution of Pichia pastorisderived recombinant proteins with mannose containing O-and N-linked glycans decreases specificity of diagnostic tests

Schistosoma mansoni-infected mice produce antibodies that cross-react with plant, insect, and mammalian glycoproteins and recognize the truncated biantennary N-glycan Man 3 GlcNAc 2 -R

Methylobacterium sp. isolated from a Finnish paper machine produces highly pyruvated galactan exopolysaccharide

Composition and desiccation-induced alterations of the cell wall in the resurrection plant Craterostigma wilmsii

Structural characteristics of pectic polysaccharides from olive fruit (Olea europaea cv moraiolo) in relation to processing for oil extraction

Protein N-glycosylation is similar in the moss Physcomitrella patens and in higher plants

Identification of the ZPC oligosaccharide ligand involved in sperm binding and the glycan structures of Xenopus laevis vitelline envelope glycoproteins

Conversion of lactose to b-D-galactopyranosyl-(1 ! 4)-D-arabinohexos-2-ulose-(2 ! dehydrolactose) and lactobiono-1,5-lactone by fungal pyranose dehydrogenase

An endorsement to create open access databases for analytical data of complex carbohydrates

Bioinformatics for glycomics: Status, methods, requirements and perspectives

Fragmentation studies of noncovalent sugar-sugar complexes by infrared atmospheric pressure MALDI

Study of peptide-sugar non-covalent complexes by infrared atmospheric pressure matrix-assisted laser desorption/ionization

Liquid infrared atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry of sialylated carbohydrates

Fragmentation of sialylated carbohydrates using infrared atmospheric pressure MALDI ion trap mass spectrometry from cation-doped liquid matrixes

Hydrophilic affinity isolation and MALDI multiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics

The (2-phenyl-2-trimethylsilyl)ethyl-(PTMSEL)-linker in the synthesis of glycopeptide partial structures of complex cell surface glycoproteins

Initiation of mucin-type O-glycosylation in Dictyostelium is homologous to the corresponding step in animals and is important for spore coat function

Site-specific characterization of the N-linked oligosaccharides of a murine immunoglobulin M by high-performance liquid chromatography/ electrospray mass spectrometry

Cloning and characterization of a novel UDP-GalNAc:polypeptide Nacetylgalactosaminyltransferase, pp-GalNAc-T14

Anti-oxidation of agar oligosaccharides produced by agarase from a marine bacterium

Crystallization of glycosylated human BACE protease domain expressed in Trichoplusia ni

MsbA transporter-dependent lipid A 1-dephosphorylation on the periplasmic surface of the inner membrane: Topography of Francisella novicida LpxE expressed in Escherichia coli

A four-component one-pot synthesis of a-gal pentasaccharide

A convergent strategy for the preparation of Nglycan core di-, tri-, and pentasaccharide thioaldoses for the sitespecific glycosylation of peptides and proteins bearing free cysteines

Site-specific glycosylation of an aglycosylated human IgG1-Fc antobody protein generates neoglycoproteins with enhanced function

Long-acting folliclestimulating hormone analogs containing N-linked glycosylation exhibited increased bioactivity compared with O-linked analogs in female rats

Silver triflate. A mild alternative catalyst for glycosylation conditions using trichloroacetimidates as glycosyl donors

Fungal b-N-acetylhexosaminidases with high b-N-acetylgalactosaminidase activity and their use for synthesis of b-GalNAc-containing oligosaccharides

Facile formation of novel carbohydrate-amino acid conjugates by reductive amination

Covalent structure of soybean seed coat peroxidase

Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications

Photoaffinity labelling of the human GM2-activator protein. Mechanistic insight into ganglioside GM2 degradation

Expression of the GM2-activator protein in the methylotrophic yeast Pichia pastoris, purification, isotopic labeling, and biophysical characterization

Ligands of the asialoglycoprotein receptor for targeted gene delivery, part 1: Synthesis of and binding studies with biotinylated cluster glycosides containing N-acetylgalactosamine

Molecular characterization and allergenic activity of Lyc e 2 (b-fructofuranosidase), a glycosylated allergen of tomato

Physicochemical characterisation of cationic polybutylcyanoacrylat-nanoparticles by fluorescence correlation spectroscopy

Carbohydrate moieties can induce mediator release: A detailed characterization of two major timothy grass pollen allergens

O-Glycosyl amino acids by 2-nitrogalactal concatenation-Synthesis of a mucin-type O-glycan

Triterpenoidal lupin saponins from the Chilean legume Lupinus oreophilus Phil

Altering the strength of lectin binding interactions and controlling the amount of lectin clustering using mannose/hydroxyl-functionalized dendrimers

Characterization of the oligosaccharides associated with the human ovarian tumor marker CA125

Synthesis of divalent b-(1 ! 6)-branched (1 ! 3)-glucohexaose and trivalent b-(1 ! 6)-branched (1 ! 3)-glucotriose

Synthesis of 6-branched cyclo-(1 ! 3)-glucohexaose and glucooctaose

Solid-phase synthesis of complex oligosaccharides using azidoglucose as a glycosyl acceptor

Localization of defined carbohydrate epitopes in bovine polysialylated NCAM

A novel GlcNAca1-HPO 3 -6Gal(1 ! 1)ceramide antigen and alkylated inositol-phosphoglycerolipids expressed by the liver fluke Fasciola hepatica

The parasitic trematode Fasciola hepatica exhibits mammalian-type glycolipids as well as Gal(b1 ! 6)Gal-terminating glycolipids that account for cestode serological cross-reactivity

Glycopeptide analysis by matrixassisted laser desorption/ionization tandem time-of-flight mass spectrometry reveals novel features of horseradish peroxidase glycosylation

A novel Gal(b1 ! 4)Gal(b1 ! 4)Fuc(a1 ! 6)-core modification attached to the proximal N-acetylglucosamine of keyhole limpet haemocyanin (KLH) N-glycans

Identification of a-galactosyl epitope mimetics through rapid generation and screening of C-linked glycopeptide library

Infrared multiphoton dissociation of alkali metalcoordinated oligosaccharides

Infrared laser isolation of ions in Fourier transform mass spectrometry

First synthesis of D-mannose penta-and decasaccharides, the repeating unit and its dimer of the cell-wall mannan of Candida kefyr IFO 0586

Comparative proteomics of glycoproteins based on lectin selection and isotope coding

Characterization of macrocyclic polysaccharides using matrix-assisted laser desorption/ionization timeof-flight mass spectrometry

Carbon nanotubes as assisted matrix for laser desorption/ionization time-of-flight mass spectrometry

Conformational analysis of chirally deuterated tunicamycin as an active site probe of UDP-N-acetylhexosamine:polyprenol-P N-acetylhexosamine-1-P translocases

Intact glycation end products containing carboxymethyl-lysine and glyoxal lysine dimer obtained from synthetic collagen model peptide

Synthesis and antigenic property of a novel sialyl 6-O-sulfo Lewis X neoglycolipid containing lactamized neuraminic acid

Convenient synthesis of a glycopeptide analogue having a complex type disialyl-undecasaccharide

Synthetic studies on glycosphingolipids from Protostomia phyla: Total syntheses ANALYSIS OF CARBOHYDRATES AND GLYCOCONJUGATES & of glycosphingolipids from the parasite, Echinococcus multilocularis

Transglycosylation reaction of Mucor hiemalis endo-b-Nacetylglucosaminidase using sugar derivatives modified at C-1 or C-2 as oligosaccharide acceptors

A novel oligosaccharide on bovine peripheral myelin glycoprotein P0

A practical synthesis of a (1 ! 6)-linked b-Dglucosamine nonasaccharide

Synthesis of biantennary b-D-(1 ! 6) glucosamine oligosaccharides

Structural determination of the polar glycoglycerolipids from thermophilic bacteria Meiothermus taiwansis

Computational estimates of the gas-phase acidities of dihydroxybenzoic acid radical cations and their corresponding neutral species

Proteomic analysis on structural proteins of severe acute respiratory syndrome coronavirus

An Nacetylglucosaminyltransferase of the Golgi apparatus of the yeast Saccharomyces cerevisiae that can modify N-linked glycans

Maltosyl-erythritol, a major transglycosylation product of erythritol by Bacillus stearothermophilus maltogenic amylase

Enzymatic synthesis of two salicin analogues by reaction of salicyl alcohol with Bacillus macerans cyclomaltodextrin glucanyltransferase and Leuconostoc mesenteroides B-742CB dextransucrase

Synthesis of GlcNAcpb-(1 ! 3)-Galp-a-(1 ! 2)-6-deoxy-altroHepp-a-(1-O-propyl), an Oantigenic repeating unit from C. jejuni O:23 and O:36

Novel efficient routes to heparin monosaccharides and disaccharides achieved via regio-and stereoselective glycosidation

Relationships between the N-glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures

Qualitative and quantitative analysis of low molecular weight compounds by ultraviolet matrix-assisted laser desorption/ionization mass spectrometry using ionic liquid matrices

Structure and biological activity of the short-chain lipopolysaccharide from Bartonella henselae ATC 49882T

Mass spectrometry of oligosaccharides

Capillary electrophoresis-mass spectrometry for glycoscreening in biomedical research

Synthesis and purity assessment of tetra-and pentaacyl lipid A of Chlamydia containing (R)-3-hydroxyicosanoic acid

Synthesis of a hexasaccharide fragment of group E streptococci polysaccharide and the tetrasaccharide repeating unit of E. coli O7: K98:H6

An efficient synthesis of a hexasaccharide-The repeating unit of the exopolysaccharide from Cryptococcus neoformans serovar A

Facile syntheses of the hexasaccharide repeating unit of the exopolysaccharide from Cryptococcus neoformans serovar A

Synthesis of a hexasaccharide, the repeating unit of O-deacetylated GXM of C. neoformans serotype A

A general method for the synthesis of oligosaccharides consisting of a-(1 ! 2)-and a-(1 ! 3)-linked rhamnan backbones and GlcNAc side chains

Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry

Gasphase potassium binding energies of MALDI matrices: An experimental and theoretical study

The Emb proteins of mycobacteria direct arabinosylation of lipoarabinomannan and arabinogalactan via an Nterminal recognition region and a C-terminal synthetic region

Rapid determination of advanced glycation end products of proteins using MALDI-TOF-MS and PERL script peptide searching algorithm

Cloning and characterization of a new human UDP-N-acetyl-a-D-galactosamine: Polypeptide N-acetylgalactosaminyltransferase, designated pp-Gal-NAc-T13, that is specifically expressed in neurons and synthesizes GalNA-serine/threonine antigen

Strategy for profiling and structure elucidation of mucin-type oligosaccharides by mass spectrometry

Profiling the morphological distribution of O-linked oligosaccharides

Hemodextrin: A self-assembled cyclodextrinporphyrin construct that binds dioxygen

N-linked oligosaccharide analysis of glycoprotein bands from isoelectric focusing gels

Synthesis of a-S-linked glycopeptides in water containing solution

Caenorhabditis elegans triple null mutant lacking UDP-N-acetyl-D-glucosamine:a-3-D-mannoside b1,2-N-acetylglucosaminyltransferase

Development of an assay system for saikosaponin a using anti-saikosaponin a monoclonal antibodies

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