key: cord-0971234-9nqdxwdk
authors: Mishra, Pratyush Kumar; Kang, Myeong-Gyun; Lee, Hakbong; Kim, Seungjoon; Choi, Subin; Sharma, Nirmali; Park, Cheol-Min; Ko, Jaewon; Lee, Changwook; Seo, Jeong Kon; Rhee, Hyun-Woo
title: A chemical tool for blue light-inducible proximity photo-crosslinking in live cells
date: 2021-12-10
journal: Chemical science
DOI: 10.1039/d1sc04871f
sha: 16b26e76636aff6000022e9991d9d16c503033e9
doc_id: 971234
cord_uid: 9nqdxwdk

We developed a proximity photo-crosslinking method (Spotlight) with a 4-azido-N-ethyl-1,8-naphthalimide (AzNP) moiety that can be converted to reactive aryl nitrene species using ambient blue light-emitting diode light. Using an AzNP-conjugated HaloTag ligand (VL1), blue light-induced photo-crosslinked products of various HaloTag-conjugated proteins of interest were detected in subcellular spaces in live cells. Chemical or heat stress-induced dynamic changes in the proteome were also detected, and photo-crosslinking in the mouse brain tissue was enabled. Using Spotlight, we further identified the host interactome of SARS-CoV-2 nucleocapsid (N) protein, which is essential for viral genome assembly. Mass analysis of the VL1-crosslinked product of N-HaloTag in HEK293T cells showed that RNA-binding proteins in stress granules were exclusively enriched in the cross-linked samples. These results tell that our method can reveal the interactome of protein of interest within a short distance in live cells.

The cross-linked EGFP-FRB and FKBP25-V5-HaloTag product is marked with a red asterisk and noncrosslinked FKBP25-V5-HaloTag (f) and EGFP-FRB (g) are marked with blue asterisks. (h) Western blot result of cross-linked FKBP25-V5-HaloTag and EGFP-FRB after immunoprecipitation of FKBP25-V5-HaloTag via its C-terminal-tagged biotinylation acceptor peptide (AP). In the cell lysate, AP was biotinylated by the addition of biotin ligase (BirA), biotin, and ATP, and biotinylated proteins were enriched by streptavidin magnetic beads (see Methods). In the eluted fraction, both the cross-linked EGFP-FRB and FKBP25-V5-HaloTag products (red asterisk) and non-crosslinked EGFP-FRB (blue asterisk) were observed in the rapamycin-treated sample. Figure S2 (Related to Figure 1 ) (a) Confocal imaging of the result in HEK293T cells. FKBP2 5-V5-HaloTag was transfected by Lipofectamine 2000 (Invitrogen) and rapamycin (100 nM) wa s treated for 1 h. FKBP25-V5-HaloTag was visualized by anti-V5 antibody and anti-mouse Alex a fluor 568 antibody after fixation and permeabilization. The green channel shows the EGFP signal. After rapamycin treatment, the nucleocytoplasmic pattern of EGFP-FRB was changed to a cytosolic localization pattern where FKBP25-V5-HaloTag is localized. Scale bar = 10 µm. (b) and (c) Ponceau stain of respective western blots in Figure 1e . (d) Western blot results of ra pamycin-induced HA-FRB cross-linking using FKBP26-HaloTag in HEK293T cells with blue LE D light; the cross-linked product is marked with a red asterisk. cells were prepared with a 100 pi dish scale for mass analysis, half of which (three plates) were incubated with VL1, illuminated under blue LED light, and lysed. From each plate, half the volume of the lysates was incubated with BirA for the in vitro biotinylation reaction of AP (or Avitag). All samples, including negative controls (i.e., no VL1 and no light/no biotin ligase), were incubated with SA-bead and SAbead eluates, trypsin-digested, and analyzed by LC-MS/MS. Proteins exclusively identified in the sample "+VL1, +BirA" were obtained after triple filtration with the results of negative controls. Following this protocol, we performed the mass sampling and LC-MS/MS analysis of N-HaloTag-AP and HaloTag-Avitag. Figure S9 ) over the VL1-crossl inked and no biotinylated N-HaloTag-AP (+VL1, -BirA; depicted as sample 3 of Figure S9 ). FC (a) Validation of protein-protein interaction by a VL-crosslinking assay. Either N-or C-terminal G3BP1-conjugated HaloTag constructs (i.e., G3BP1-HaloTag, HaloTag-G3BP1) were co-expressed with G3BP1 in HEK293T cells, and either VL1 or VL2 was incubated at 10 μM for 1 h. No probe-treated samples were included as negative controls. All samples were illuminated by blue LED light (10 min) and lysed for western blot analysis using anti-V5 antibody (left) and anti-GFP antibody (right). In the result of the anti-V5 western blot, both VL1 and VL2 generated cross-linked products of G3BP1-HaloTag and HaloTag-G3BP1 proteins. However, in the anti-GFP western blot result, only VL1 generated the N-GFP cross-linked product on the HaloTag-G3BP1 protein whose HaloTag is conjugated at the N-terminus of G3BP1. (b) Either a short linker (11 aa) or long linker (33 aa) construct of HaloTag-G3BP1 (i.e., HaloTag-11aa-G3BP1 HaloTag-33aa-G3BP1) was co-expressed with N-GFP in HEK293T cells, and incubated with either VL1 or VL2 at 10 μM for 1 h. No probe-treated samples were included as negative controls. All of the samples were illuminated by blue LED light (10 min) and lysed for western blot analysis using anti-V5 antibody (left) and anti-GFP antibody (right). In the result of anti-V5 western blot, both VL1 and VL2 generated cross-linked products of HaloTag-11aa-G3BP1 and HaloTag-33aa-G3BP1 proteins. In the anti-GFP western blot result, both HaloTag-11aa-G3BP1 and HaloTag-33aa-G3BP1 showed VL1-mediated N-GFP cross-linked products. Photo-crosslinked products with VL1 are marked with red asterisks and non-crosslinked HaloTag-V5-AP proteins are marked with blue asterisks. 

Plasmids and Cloning. Genes were cloned into the specified vectors using standard enzymat ic restriction digest and ligation with T4 DNA ligase. To generate constructs where short tags (e.g., V5 epitope or AviTag) or signal sequences were appended to the protein, the tag was in cluded in the primers used to PCR-amplify the gene. PCR products were digested with restric tion enzymes and ligated into cut vectors (e.g., pcDNA3, pCDNA5, pDisplay, pET21a and pH6 HTN). In all cases, the CMV promoter was used for expression in mammalian cells. Table S1 below summarizes the genetic constructs cloned and used for this study.

Protein purification. Gene encoding protein was amplified using PCR and cloned into modif ied pET21a or pH6HTN vector with histidine tag (6X His). The genes (FKBP12-EGFP, FRB-V5-HaloTag, and BirA-His6 in Table S1 ) were transformed into expressed in BL21 (DE3) Escherich ia coli cells following induction at 42ºC for 30 sec and BL21 was cultured on ampicillin treate d agar plate. A colony was picked into 5ml ampicillin containing LB-Broth overnight, and 1ml LB was transferred to 1L new LB broth. After reaching to 0.5 optical density, cells were treat ed with 0.25 mM IPTG and cultured at 18 ºC for 24 hr. Cells were harvested 24 hours post-i nduction, lysed by B-per buffer (Invitrogen), centrifugation, and protein was purified by Ni 2+ -NTA chromatography. The eluted protein was finally concentrated, and free imidazole ring wa s removed by using Amicon Ultra-15 centrifugal filter (Mw. 10 kDa cutoff, Millipore) and flash -frozen in liquid nitrogen for storage. For crystallization, HaloTag proteins were purified as de scribed previously 1 . For the formation of HaloTag complexed with VL1 and UL2 ligands, purifie d proteins were mixed with 3-fold molar excess of VL1 and UL2, respectively. After incubating for 3 hours on ice, proteins were injected onto a size-exclusion column (GE healthcare, supe rdex200 16/600) equilibrated with 25 mM Tris pH 7.5, 150 mM NaCl, 5 mM DTT. Finally, the eluted proteins were concentrated to 16 mg/ml and stored at -80℃.

Crystal structure determination. The Halotag proteins bound to VL1 or UL2 were crystalliz ed by the hanging-drop method by mixing 1 μl protein and 1 μl of crystallization solution. T he crystallization condition of the HaloTag-VL1 complex was 1.2 M Ammonium sulfate, 0.1 M citric acid pH 5.3, and 1% MPD at room temperature, and HaloTag-UL2 complex were crystal lized under buffer containing 25% PEG 8K, 0.1 M MES pH 6.5, 0.2 M Na-acetate at room te mperature. The crystals were soaked into a cryoprotectant solution consisting of crystallization buffer plus 30% glycerol, and flash frozen in liquid nitrogen. X-ray diffraction data were coll ected at the beamline 7A of the Pohang Accelerator Laboratory (PAL), and processed by HKL 2000 program 2 . The crystal structure was solved by the molecular replacement by Phenix 3 usi ng apo-HaloTag (PDB accession code: 5Y2X) as a search model. Model building and refineme nt were carried out using Coot 4 and Phenix 3 , respectively. Data collection and refinement stat istics are summarized in Table S2 .

For transfection cells were cultured in DMEM (hyclone, SH30243) supplemented with 10% FBS, 2 mM L-glutamine, 50 units/mL penicillin, and 50 µg/ml streptomycin at 37°C under 5% CO2, for 12 well plate at 60-70% confluency, 1000 ng plasmid DNA was mixed with 2μg of Polyethylenimine (PEI, Polysciences, 23966) using 100 uL no-FBS, DMEM and added to the well, after 2-3 hours of addition, media was changed to full media described above and transfected cells were used for imaging or crosslinking experiments after 20-24 hrs of transfection.

General Imaging Protocol. For imaging experiments, HEK-AD or HEK293T cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 50 units/mL penicillin, and 50 µg/ml streptomycin at 37°C under 5% CO2. After 24 hr transfection, 10µM VL1 in DMEM was treated into cells for 1 hr. Then, cells were washed by DPBS (thermofisher, 21300) 3 times and cells were fixed with 4% paraformaldehyde solution (chembio, CBPF-9004) in DPBS at room temperature for 15 min. Cells were then washed with DPBS two times and permeabilized with chilled methanol at -20°C for 5 min. Cells were washed again two times with DPBS and blocked for 30 min with 2% BSA (milipore, 82-100-6) in DPBS ("blocking buffer") at room temperature. To detect HaloTag fusion protein expression, cells were incubated with mouse anti-V5 antibody (Invitrogen, cat. no. R960-25, 1:5000 dilution) for 1 h at room temperature. After washing four times with TBST each 5 min, cells were simultaneously incubated with secondary Alexa Fluor 568-goat anti-mouse IgG (Invitrogen, cat. no. A-11004, 1:1000 dilution) for 30 min at room temperature.

General Protocol for Visualizing Crosslinking on Western blot. For crosslinking experiments, HEK-293T cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 50 units/mL penicillin, and 50 µg/ml streptomycin at 37°C under 5% CO2. After 24 hr transfection, 10µM VL1 in DMEM was treated into cells for 1 hr. Then, cells were washed by DPBS 3 times and subjected to Blue LED light for 10 min and RIPA lysis buffer (Elpis biotech, EBA-1149) was added after removal of DPBS. Lysis was performed for 30 min at 4 o C Then, sample was loaded into 6% SDS-PAGE gel and run at 150V for 60 min. After separation, proteins on gel was transferred to nitrocellulose membrane at 400 mA for 90 min. Protein loading level was checked by ponceau staining and ponceau was removed by 1xTBST buffer. Blocking was performed with 2% skim milk in TBST for 1 hr. Blocking solution was replaced with primary antibody in 2% skim milk and incubated for 1 hr. After 4 times (each for 5 min) of 1xTBST buffer washing, membrane was incubated with secondary antibody in 2% skim milk in TBST for 30 min. After 4 times washing with 1xTBST buffer, developing was performed with ECL kit (Biorad, 1705061) and image was taken by Gel doc machine (Genesys)

Adenoassociated virus (AAV)vector encoding mouse PSD95-V5-Halotag was generated by amplification of the full-length region (Genbank accession number: NM_019621.1) by PCR and subsequently subcloned into the pAAV-T2A-tdTomato vector (a gift from Dr. Hailan Hu, Zhejiang University, China) at XbaI and BamHI sites. 5 AAV packing plasmids (pAAV-PSD95-T2A-tdTomato, pHelper and AAV1.0 serotype 2/9) were transfected into HEK 293T cells. 72-108 hours after transfection, cells were harvested by addition of 0.5 M EDTA to the media and collected by centrifugation. These cells were subjected to four freeze-thaw cycles (7 minutes in ethanol/dry ice and 5 minutes in 37°C water) and centrifuged to divide supernatants from lysates. Collected supernatants were mixed and incubated with a solution containing 40% polyethylene glycol and 2.5 M NaCl on ice for 1 hour and centrifuged at 2000 rcf for 30 minutes. The pellets were resuspended in HEPES buffer (20 mM HEPES pH 8.0, 115 mM NaCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 2.4 mM KH2PO4, and then mixed with chloroform by vortexing for 2 minutes and centrifuged at 400 rcf for 10 minutes. The supernatant was transferred into the Amicon Ultra Centrifugal Filters (0.5 ml, 3K MWCO; Millipore) to concentrate the viruses. AAV virus titer was determined by RT-PCR, and the virus concentrated at 1 X 1010-1011 infectious units/ml was used in the experiments. 4-week-old C57BL/6N male mice were anesthetized by intraperitoneal injection of avertin solution (2% 2,2,2-tribromoethanol dissolved in tert-amylalcohol), and fixed in a stereotaxic apparatus. Viruses were injected into hippocampus of mice with a Hamilton syringe using a Nanoliter 2010 Injector (World Precision Instruments) as following coordination: anteroposterior (AP), -2.1 mm; medial-lateral (ML), ± 1.2 mm; and dorsal-ventral (DV), 2.2 mm from bregma. After surgery, injected mouse was returned to its home cage for recovery. Hippocampi dissected from brains of injected mice were minced and incubated in 10 μM of VL1 for an hour. Tissues were washed by PBS twice and then, irradiated to Blue LED light for 30 min. After crosslinking with VL1, dissected hippocampi were homogenized in ice-cold homogenization buffer (320 mM sucrose, 5 mM HEPES-NaOH (pH 7.5), 1 mM EDTA, 0.2 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 mM Na3VO4.) The homogenized sample was centrifuged at 1000 x g for 10 minutes, and the supernatant was centrifuged at 15,000 x g for 20 minutes. The pellets were resuspended in RIPA lysis buffer and centrifuged at 15,000 x g for 10 minutes. After concentration measurement by BCA assay (thermofisher, 23225), immunoblotting was performed as described above.

Mass sample preparation of photo-crosslinked N-HaloTag For the mass analysis of the crosslinked product HEK293T cells were grown as triplicate in 100π dish until confluency was 60-70% then 8,000 ng of plasmid DNA was transfected using PEI transfection reagent and media was changed to full media after 3 hours, after 24 hours of transfection 10 μM VL1 was incubated for 1 hour and washed 3 times with cold DPBS before crosslinking in Blue LED for 10 min, cells were scrapped in DPBS made pellet and lysed by RIPA buffer, then lysate was subjected to in-vitro biotinylation using recombinant wild type BirA solution (10 μM BirA, 10μM biotin, 200μM ATP, and 500μM MgCl2) for 12 hours at room temperature. For removing free biotin, lysates were loaded on amicon filter and centrifugated at 12,000 x g for 4 x 15 min. 1xTBS buffer was added into concentrated lysate up to 400 μL and then, 100 μL of washed streptavidin beads were added. After 10 min incubation, 2ml SDS in TBS was added up to 2~10% final concentration and incubated for an hour. Beads were washed by 2% SDS in TBS buffer 4 times and incubated with 10mM DTT in 50mM ABC buffer at 37℃ for an hour. Alkylation was performed by 55mM IAM in ABC buffer at 37℃ in dark condition. After an hour incubation, solution was replaced by 200 μL of trypsin solution (2 μg trypsin, 1mM CaCl2 in 50mM ABC buffer) and mixed at 37℃ for 12 hours. Then, supernatant was desalted using zip-tip (thermofisher, 87784), eluted fractions were dried using speed vac and kept in deep freezer until loaded on LC-MS/MS.

Peptides were analyzed by Thermo-Scientific® Q Exactive Plus equipped with a nanoelectrospray ion source. A C18 reverse-phase HPLC column (500 mm × 75 µm i.d.) was used to separate the peptide mixture using a 2.4-17.6% acetonitrile/0.1% formic acid gradient for 120 min at a flow rate of 300 nL/min. For MS/MS analysis, precursor ion scan MS spectra (m/z 350 -2000) were acquired using the Orbitrap spectrometer at a resolution of 70 K at 200 m/z with an internal lock mass. Resolution of 17,500 at m/z 200 for HCD spectra was set and the 15 most intensive ions were isolated and fragmented by higher energy collisional dissociation (HCD).

For protein identification, Scaffold (version 4.11.0, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater t han 99.0% probability and contained at least 2 identified peptide. Protein probabilities were assigned by the Protein Prophet algorithm. 6 Proteins that contained similar peptides and cou ld not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.

In database searching, tandem mass spectra were extracted by Proteome Discoverer (version 2.2, Thermo Fisher Scientific, San Jose, CA). All MS/MS samples were analyzed using Sequest (XCorr Only). Sequest was set up to search Homo sapiens protein sequence database (42230 e ntries, UniProt (http://www.uniprot.org/)) assuming digestion with trypsin. Sequest was search ed with a fragment ion mass tolerance of 0.80 Da and a parent ion tolerance of 10.0 PPM. C arbamidomethyl of cysteine was specified in Sequest as a fixed modification. Oxidation of me thionine and acetyl of the n-terminus were specified in Sequest as variable modifications. For the volcano plot analysis (Figure 5d, Figure S10a,b) , five multiple imputation of missing value s were followed via panda-view software 7 (see Supplementary Dataset 1).

Scheme S1. Synthetic Scheme of UL1-UL4 and VL1-VL2.

To a solution of 2-(2-(2azidoethoxy)ethoxy)ethanol (17.5 mg, 0.1mmol) 8 in DMF at 0 °C was added NaH (6 mg, 0.15 mmol). After stirring for 30 minutes at 0 °C, 1-chloro-6-iodohexane (18.6 μL, 0.12 mmol) was added. The mixture was stirred for 1h, and warmed to r.t., and stirred for 2 h. Ammonium chloride was introduced at 0 °C into the reaction mixture, extracted with diethyl ether (10 mL × 3), washed with saturated brine (10 mL), dried over anhydrous Na2SO4 and filtered, and concentrated in vacuo. The crude material was purified by flash chromatography on silica gel to afford compound 1b (12 mg, yield : 40%). 1 

After two vacuum/H2 cycles to replace air inside the reaction tube with hydrogen, the mixture of 1b (35 mg, 0.12 mmol),10% Pd/C (3.5 mg 10 wt % of 1b) and 2-3 drops of 1N HCl in MeOH (0.6 mL) was stirred at r.t. under hydrogen balloon S32 for 2 h. The reaction mixture was filtered using celite filter to afford compound 1c as quantitative yield. 1 H NMR (400 MHz, DMSO-d6) δ 8.39 -8.02 (m, 3H), 3.59 (dq, J = 6.7, 3.6, 3.1 Hz, 4H), 3.55 -3.41 (m, 8H), 3.35 (d, J = 2.9 Hz, 2H), 2.90 (t, J = 5.3 Hz, 2H), 1.73 -1.63 (m, 2H), 1.46 (dd, J = 9.9, 4.7 Hz, 2H), 1. 

To synthesize aryl-azide-PEG2 HaloTag Ligand (UL1), 4-azidobezoic acid 5 (83 mg, 0.50 mmoles) was taken in 10 ml round bottom flask with HATU (214 mg, 0.56 mmoles), DIPEA (131.3 mg, 1.02 mmoles) and dissolved in 2 ml of DCM. The reaction mixture was stirred at room temperature for 30 minutes or until the color turned brown, then 2-(2-((6-chlorohexyl)oxy)ethoxy)ethanamine (138 mg, 0.61 mmoles) 9 dissolved in 1 ml DCM was added dropwise to reaction mixture and reaction was stirred for additional 3 hours at room temperature. After completion of the reaction, solvent was removed under reduced pressure to obtain a colorless oil which was purified by column chromatography over silica gel (Eluent: 40 % EtOAc in Hexane) to furnish the pure 6 (168 mg, 90%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.80 (d, J = 8.7 Hz, 2H), 7.06 (d, J = 8.7 Hz, 2H), 6.67 (br s, 1H), 3.70 -3.63 (m, 6H), 3.62 -3.57 (m, 2H), 3.52 (t, J = 6.7 Hz, 2H), 3.46 (t, J = 6.7 Hz, 2H), 1. 

To synthesize aryl-azide-PEG3 HaloTag Ligand (UL2), 4-azidobezoic acid 5 (45 mg, 0.28 mmoles) was taken in 10 ml round bottom flask with HATU (105 mg, 0.28 mmoles), DIPEA (64 mg, 0.58 mmoles) and dissolved in 2 ml of DCM. The reaction mixture was stirred at room temperature for 30 minutes or until the color turned brown, then 2-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethoxy)ethanamine (68mg, 0.25 mmoles) 10 dissolved in 1 ml DCM was added dropwise to reaction mixture and reaction was stirred for additional 3 hours at room temperature. After completion of the reaction, solvent was removed under reduced pressure to obtain a colorless oil which was purified by column chromatography over silica gel (Eluent: 60 % EtOAc in Hexane) to furnish the pure 14 (98 mg, 95%) as a colorless oil. , 1H NMR (400 MHz, Acetone) δ 7.95 (d, J = 8. 

To synthesize N-(2-(2-(2-((6-chlorohexyl)oxy)ethoxy)ethoxy)ethyl)-4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamide (UL4), a solution of 4-(3-(trifluoromethyl)-3H-diazirin-3yl)benzoic acid (11.5 mg, 0.05 mmol), EDC-HCl (10.5 mg, 0.055 mmol), TEA (7 uL, 0.05 mmol) and DMAP

582 mmol) was taken in a 10 ml round bottom flask and 1 ml DMF with HATU (254 mg, 0.67 mmol) and DIPEA (240 ul, 1.3 mmol) and stirred at room temperature for 30 min until reaction mixture turned to yellowish precipitate then NH2-PEG2-HTL was added along with 1 ml DMF and reaction mixture turns uniform liquid for a moment and again becomes precipitate kept on stirring for another 1 hour and then work up was performed using EtOAc and water, organic layer dried over Na2SO4 and purification was performed using silica gel Colum (1% MeOH in CHCl3) to afford 180 mg of yellow powder(60%)

Synthesis of VL2 3-(6-azido-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propanoic acid(116 mg, 0.37 mmol) was taken in a 10 ml round bottom flask and 1 ml DMF with HATU (160 mg, 0.42 mmol) and DIPEA (255 ul,) and stirred at room temperature for 30 min until reaction mixture turned to yellowish precipitate then NH2-PEG3-HTL was added along with 1 ml DMF and reaction mixture turns uniform liquid for a moment and again becomes precipitate kept on stirring for another 1 hour and then work up was performed using EtOAc and water, organic layer dried over Na2SO4 and purification was performed using silica gel Colum

Architecture Mapping of the Inner Mitochondrial Membrane Proteome by Chemical Tools in Live Cells

Processing of X-ray diffraction data collected in oscillation mode

PHENIX: a comprehensive Python-based system for macromolecular structure solution

Features and development of Coot

Npas4 regulates IQSEC3 expression in hippocampal somatostatin interneurons to mediate anxiety-like behavior

A Statistical Model for Identifying Proteins by Tandem Mass Spectrometry

PANDA-view: an easy-to-use tool for statistical analysis and visualization of quantitative proteomics data

A vimentin binding small molecule leads to mitotic disruption in mesenchymal cancers

Small-molecule hydrophobic tagging-induced degradation of HaloTag fusion proteins

Direct pH measurements by using subcellular targeting of 5(and 6-) carboxyseminaphthorhodafluor in mammalian cells

Base-Mediated One-Pot Synthesis of Aliphatic Diazirines for Photoaffinity Labeling