key: cord-0905597-28rbae2k authors: Gajbhiye, A.; Nalbant, A.; Heunis, T.; Sidgwick, F.; Porter, A.; Taha, Y.; Trost, M. title: A fast and sensitive absolute quantification assay for the detection of SARS-CoV-2 peptides using Parallel Reaction Monitoring Mass Spectrometry date: 2022-03-21 journal: nan DOI: 10.1101/2022.03.18.22272462 sha: 9d4bcc8b6f8cda8e849f37a8f7ce259704f81f02 doc_id: 905597 cord_uid: 28rbae2k The on-going SARS-CoV-2 (COVID-19) pandemic has called for an urgent need for rapid and high-throughput methods for mass testing for early detection, prevention and surveillance of the disease. Here, we tested if targeted parallel reaction monitoring (PRM) quantification using high resolution Orbitrap instruments can provide the sensitivity and speed required for a high-throughput method that could be used for clinical diagnosis. Here we report a high-throughput and sensitive PRM-MS assay that enables absolute quantification of SARS-CoV-2 nucleocapsid peptides with short turn-around times. Concatenated peptides (QconCAT) synthesized using isotopically labelled SARS-CoV-2 were used for absolute quantification. We developed a fast and high-throughput S-trap-based sample preparation method, which was then successfully utilized for testing 25 positive and 25 negative heat-inactivated nasopharyngeal swab samples for SARS-CoV-2 detection. The method was able to differentiate between negative and positive patients accurately within its limits of detection. Moreover, extrapolating from the QconCAT absolute quantification, our data show that patients with Ct values as low as 17.5 have NCAP protein amounts of around 7.5 pmol in swab samples. The present high-throughput method could potentially be utilized in specialized clinics as an alternative tool for detection of SARS-CoV-2. 100 µg/mL gentamicin and 0.5 µg/mL amphotericin B, as recommended by the CDC 1 6 3 [22].The virus was inactivated by heating at 80°C for 5 min and 1 mL of 20% SDS was 1 6 4 added to the VTM containing the swab such that the total SDS concentration was ~5 % which 1 6 5 is compatible with the S-trap method of sample digestion. The samples were vortexed for 15 1 6 6 min followed by centrifugation at 1500 x g and the entire solution was collected, aliquoted 1 6 7 and stored at -80°C for further use. 1 6 8 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) RESULTS 1 6 9 1 7 0 A high-throughput LC-MS method should be reproducible, robust and sensitive. Towards our 1 7 1 goal of developing a high-throughput PRM method for detection of SARS-Cov-2 peptides, 1 7 2 we developed a fast and reproducible sample preparation method which can be used in 1 7 3 conjunction with the PRM method. The details of the of the sample preparation and LC-MS 1 7 4 method are listed below. 1 7 5 1 7 6 Sample preparation for high-throughput analysis of SARS-CoV-2 samples 1 7 7 The sample preparation was designed to accommodate nasopharyngeal swabs which are the 1 7 8 preferred sample collection methods in use in the clinic. In order to return clinical data 1 7 9 quickly, we aimed to design the sample preparation method for high-throughput sample is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 21, 2022. an EvoSep LC system and analysed on a QExactive HF mass spectrometer. The present 1 8 8 method allows efficient digestion of multiple samples with various matrices under 2 hours, 1 8 9 permitting the LC-MS and data analysis in approximately 15 mins. 1 9 0 1 9 1 Recombinant SARS-CoV-2 SPIKE and NCAP proteins were spiked at 100 pmol/mL into 1 9 2 artificial saliva or into saliva and swab samples (stored in 3 mL viral transfer medium) taken 1 9 3 from a healthy volunteer. As clinical samples will require virus inactivation, we tested 70 % 1 9 4 ethanol, heat inactivation at 80°C for 5 min as well as addition of 20% SDS to a final 1 9 5 concentration of 5% followed by heat inactivation at 80°C. After addition of ethanol or SDS, 1 9 6 the samples were shaken for 15 mins and centrifuged for 5 mins at 1500 x g. Samples were 1 9 7 further processed using 96-well S-trap plates as this workflow provides a fast method for 1 9 8 digestion of proteins in most buffers without the need for precipitation, thereby avoiding 1 9 9 sample loss and long sample preparation times. As none of the previously reported 2 0 0 proteotypic peptides of NCAP or SPIKE contain a cysteine, we tested if reduction with TCEP 2 0 1 and alkylation with IAA were necessary for the efficient detection of the target peptides. 2 0 2 Digestion efficiency was assessed by LC-MS/MS analysis of tryptic peptides and was found 2 0 3 to be unaffected even when samples were not reduced and alkylated before digestion. In the standard S-Trap protocol peptides are eluted with two aqueous (50 mM TEAB and 2 0 6 0.2% formic acid) and an organic (50% MeCN, 0.2% formic acid) step. Due to the organic 2 0 7 solvent, the samples require vacuum drying before LC-MS analysis. In order to save time, we 2 0 8 tested if we could omit the elution in MeCN, thereby allowing straight injection of the eluted 2 0 9 digests into the mass spectrometer. Indeed, omission of the organic elution did not affect 2 1 0 overall intensities of SPIKE and NCAP peptides (Supplementary Figure 1B) . Identification of SARS-CoV-2 proteotypic peptides for targeted analysis 2 1 2 Selection of appropriate, proteotypic peptides is a key step towards developing a robust and 2 1 3 reproducible targeted LC-MS assay. In order to identify the proteotypic peptides of SARS-2 1 4 CoV-2 SPIKE and NCAP proteins, recombinant proteins were digested in neat (0.1% formic 2 1 5 acid) and spiked into artificial saliva or into saliva and swab samples taken from a healthy 2 1 6 volunteer. Digests were analysed on a Q-Exactive HF in data-dependent analysis (DDA) 2 1 7 mode coupled to an Evosep LC -MS system, using the 60 sample per day (60 SPD) method 2 1 8 (Figure 2) . The DDA-based analysis of neat SPIKE and NCAP recombinant proteins 2 1 9 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) Thermo QE-HF mass spectrometer. Data was analysed by Skyline to assess the sensitivity of the 2 3 3 system as well as to validate using patient swab samples. In order to select optimal proteotypic peptides for the targeted PRM analysis, we followed 2 3 5 published selection criteria for a targeted-based LC-MS assays such as tryptic peptide length 2 3 6 of 8 to 25 amino acid and excluded peptides with possible modifications such as oxidation on 2 3 7 Methionine, deamidation on Asparagine followed by Glycine, and any other possible post-2 3 8 translational modifications [24] . Following these criteria, an initial list of tryptic peptides was 2 3 9 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 21, 2022. to run samples back-to-back without carryover due to the tip-based sample injection system, 2 5 7 which serves as a single-use trap column thereby increasing the overall life of the column, 2 5 8 making the system well-suited for a high-throughput method [26] . The PRM method was 2 5 9 further optimized for collision energies (Supplementary Figure 2) for the individually 2 6 0 selected peptides along with their corresponding SIL peptides emerging from the QconCAT. The QconCAT sequence information can be found in (Supplementary Data 4) . The 2 6 2 normalized collision energies (NCE) for the targeted peptides were optimized within the 2 6 3 range of 20-29 for all the peptides. The optimal collision energies for individual peptides 2 6 4 were selected based on the intensities of their product ions using Skyline. Details of the seven 2 6 5 peptides and collision energies used for the final PRM assay are listed in Table 1 . . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The next step in establishing the high throughput PRM method involved its assessment for 2 7 2 sensitivity and specificity. Calibration curves of the target peptides in neat, artificial saliva as 2 7 3 well as real saliva and swab samples were generated using the tryptic digests for recombinant 2 7 4 protein standards for NCAP. Recombinant tryptic peptides in 0.1% formic acid ("neat") 2 7 5 ( Figure 3A) , spiked swab ( Figure 3B ), spiked oral fluid ( Figure 3C ) and spiked saliva 2 7 6 ( Figure 3D ) were injected in a dilution series from 50 femtomoles to 50 attomoles was on 2 7 7 column. The limit of detection (LOD) for NCAP peptides was found to be as low as 170 amol 2 7 8 on column, while the limit of quantitation (LOQ) was found to be 850 amol when injected 2 7 9 neat ( Table 2) . The calibration curves for the logged intensities also depict the linearity of the 2 8 0 curves as seen in (Supplementary Figure 3) . The LOD and LOQ were calculated using the 2 8 1 formulae 3*Sa/b and 10*Sa/b respectively, where Sa is standard deviation of the calibration 2 8 2 curve response and b is the slope of the curve. The LOD and LOQ decreased to 0.63 fmol and 2 8 3 3.24 fmol on column, respectively, for the same peptide when injected as a spike-in in the 2 8 4 real saliva and swab samples. The drop in sensitivity was expected when peptide standards 2 8 5 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The existing PRM assay system needed to not only be sensitive but also robust to 2 9 0 carryout high-throughput analysis of many samples. To test the robustness of the LC-MS 2 9 1 system a continuous 200 sample test was performed with a saliva sample spiked-in with 2 9 2 NCAP peptides. The peptide AYNVTQAFGR was monitored for its product ion areas and 2 9 3 retention time. The test revealed that the system performed consistently without a notable 2 9 4 drop in sensitivity across all 200 samples, solidifying the stability of the system to handle 2 9 5 such a load (Figure 4A and 4B). 2 9 6 2 9 7 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 21, 2022. ; https://doi.org/10.1101/2022.03.18.22272462 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 21, 2022. Validation of PRM Assay using 50 patient samples 3 1 7 The final PRM method was validated using 25 positive and 25 negative patient samples. The The samples were processed according to the optimized sample preparation protocol. BCA 3 2 5 estimation of the samples revealed that the total concentration of the protein in the extraction 3 2 6 buffer ranged between 3-5 mg/mL. Out of the total sample extracted from swab in 3 mL of 3 2 7 VTM with 5% SDS buffer, only 50 μL sample (~200 μg) was taken for processing. ~10 μg 3 2 8 sample was injected on column, where concentration of QconCAT in the sample was 10 fmol 3 2 9 on column. Thus, ~1/1200 th of the total sample was used for detection of the SARS-CoV-2 3 3 0 peptides using the present method. The samples were attributed as positive only if they passed the following criteria: (i) At least 3 3 2 two out of maximum six fragment ion transitions were detectable and (ii) The retention times 3 3 3 matched that of the heavy QconCAT counterpart of the same peptide. The peptides that were 3 3 4 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 21, 2022. ; https://doi.org/10.1101/2022.03.18.22272462 doi: medRxiv preprint assigned as positive were then used for calculation of the probable absolute concentration 3 3 5 from the total H/L ratios calculated by Skyline software. 3 3 6 Our PRM method was able to detect nine positive cases, three shown here and the 3 3 7 other six in (Supplementary Figure 4) , out of the 25 positive samples correctly (Figure 5 with SAMBA II and Lumira, respectively, which were also confirmed with our method as positives. D) is a negative patient sample. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted March 21, 2022. ; https://doi.org/10.1101/2022.03.18.22272462 doi: medRxiv preprint 3 4 9 Discussion 3 5 0 The presented MS-based assay aims to directly detect viral peptides in nasopharyngeal swab 3 5 1 samples with faster processing times in a high-throughput manner. The detection is pursued 3 5 2 from miniscule amounts of total samples unlike the nucleic acid-based strategies which utilise 3 5 3 amplification. This is one of the major reasons attributing to the lower sensitivity of the 3 5 4 present MS-based targeted assay compared to nucleic acid amplification-based methods such 3 5 5 as PCR and SAMBA. While Lumira does not require amplification, it requires antibodies 3 5 6 which can be non-specific and can lead to false positives. In order to achieve more sensitivity 3 5 7 higher sample volume needs to be injected onto the system, which poses a challenge when 3 5 8 using a nano-flow LC system. Additionally, to provide a boost in sensitivity pre-enrichment methods such as SISCAPA 3 6 0 [30], aptamers [31] or bead-based enrichments as utilized by Renuse et.al [15] could be 3 6 1 incorporated in the sample preparation workflow, as the viral peptides have very low 3 6 2 abundance when measured against a background of human saliva samples rich with high-3 6 3 abundant proteins like Lys C and Amylase. The other reason that contributes towards lowered 3 6 4 detection sensitivity of the PRM assay could be assigned to the way the samples were 3 6 5 collected and stored. In many of the samples, the traces of food particles and phlegm from 3 6 6 individual patients were found, which made sample processing problematic. This highlights 3 6 7 the fact that sample collection and storage for MS-based assays should be standardized, such 3 6 8 as making sure the nasopharyngeal swab samples are taken by qualified staff, making sure 3 6 9 the swabs touch only the back of the throat and the base of the tonsils avoiding any 3 7 0 contamination. Also, efforts should be taken towards immediate processing of the samples 3 7 1 after collection or stored at -20°C to reduce any protein degradation. Conflict of interest: None. 4 3 0 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 21, 2022. ; A mass spectrometry-based targeted assay for detection of SARS-4 9 CoV-2 antigen from clinical specimens. EBioMedicine proteins. Indeed, more rigorous studies with a greater number of patient samples with 3 9 0 standardized sampling protocols are needed to establish a strong peptide concentration-viral 3 9 1 particles correlation baseline. Nonetheless, the quantification of these peptides in the 3 9 2 nasopharyngeal samples is a step in the right direction to assess not only the prognostic 3 9 3 potential of the method but also determining the concentration range of the protein in 3 9 4 correlation with the severity of the SARS-CoV-2 disease, which may prove helpful in 3 9 5 designing treatments for disease management and thereby reducing complications and 3 9 6 hopefully fatalities. 3 9 7Future Perspectives:The global response to the Covid-19 pandemic has come a long way since its inception in late 3 9 9 2019. Lately, the death toll and the number of SARS-CoV-2 variants arising with the on-4 0 0