Specific and Rapid SARS-CoV-2 Identification Based on LC-MS/MS Analysis

Schuster, Ofir; Zvi, Anat; Rosen, Osnat; Achdout, Hagit; Ben-Shmuel, Amir; Shifman, Ohad; Yitzhaki, Shmuel; Laskar, Orly; Feldberg, Liron

doi:10.26434/chemrxiv.13055873.v1

Cited by 2 publications

(3 citation statements)

References 15 publications

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“…The detection of this peptide was shared with other studies to compare the concordance of the MS method with the current gold standard assay on swab samples previously tested as positive by positive quantitative reverse transcription PCR (RT-qPCR). 20 A high percentage of N protein sequence peptides were similarly monitored as reported by others, 7,16,20,21 while fewer authors monitored other proteins besides S and N proteins in real samples. In fact, lower sequence coverage has been reported in full scan experiments for the small membrane protein envelope (E), membrane protein (M), or ORF proteins in virus-enriched cells.…”

Section: ■ Results and Discussionmentioning

confidence: 67%

Identification of SARS-CoV-2 Proteins from Nasopharyngeal Swabs Probed by Multiple Reaction Monitoring Tandem Mass Spectrometry

et al. 2021

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Numerous reverse transcription polymerase chain reaction (RT-PCR) tests have emerged over the past year as the gold standard for detecting millions of cases of SARS-CoV-2 reported daily worldwide. However, problems with critical shortages of key reagents such as PCR primers and RNA extraction kits and unpredictable test reliability related to high viral replication cycles have triggered the need for alternative methodologies to PCR to detect specific COVID-19 proteins. Several authors have developed methods based on liquid chromatography with tandem mass spectrometry (LC–MS/MS) to confirm the potential of the technique to detect two major proteins, the spike and the nucleoprotein, of COVID-19. In the present work, an S-Trap mini spin column digestion protocol was used for sample preparation prodromal to LC–MS/MS analysis in multiple reactions monitoring ion mode (MRM) to obtain a comprehensive method capable of detecting different viral proteins. The developed method was applied to n. 81 oro/nasopharyngeal swabs submitted in parallel to quantitative reverse transcription PCR (RT-qPCR) assays to detect RdRP, the S and N genes specific for COVID-19, and the E gene for all Sarbecoviruses , including SARS-CoV-2 (with cycle negativity threshold set to 40). A total of 23 peptides representative of the six specific viral proteins were detected in the monitoring of 128 transitions found to have good ionic currents extracted in clinical samples that reacted differently to the PCR assay. The best instrumental response came from the FLPFQFGR sequence of spike [558−566] peptide used to test the analytical performance of the method that has good sensitivity with a low false-negative rate. Transition monitoring using a targeted MS approach has the great potential to detect the fragmentation reactions of any peptide molecularly defined by a specific amino acid sequence, offering the extensibility of the approach to any viral sequence including derived variants and thus providing insights into the development of new types of clinical diagnostics.

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Section: ■ Results and Discussionmentioning

confidence: 67%

Identification of SARS-CoV-2 Proteins from Nasopharyngeal Swabs Probed by Multiple Reaction Monitoring Tandem Mass Spectrometry

et al. 2021

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“…33 Subsequently, Feldberg's group detected HTPINLVR in nasopharyngeal swabs with RT-PCR validated SARS-CoV-2 infections using 20 min LC−MS/MS. 24 The spike protein amount in clinical setting can be estimated as follows: individual virions contain ∼228 spike proteins per virion, which is the average of the estimated from each virion containing 25−127 spike protein trimers. 51 Multiplying a moderate virion/mL of 10 7 by 228 yields 2.3 × 10 8 spike protein molecules/mL or 3,800 amol/ mL.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…As proteomics lacks an amplification strategy similar to PCR, efficient recovery of viral proteins is critical. Sample preparation methods reported include precipitation, 24,25 and solid-phase extraction. 26,27 Both selective, e.g., antibody based extraction of N-protein, 28 and nonselective, e.g., hydrophilic interaction chromatography (HILIC) extraction targeting total protein have been reported.…”

Section: ■ Introductionmentioning

confidence: 99%

Evaluation of Variant-Specific Peptides for Detection of SARS-CoV-2 Variants of Concern

et al. 2022

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The SARS-CoV-2 omicron variant presented significant challenges to the global effort to counter the pandemic. SARS-CoV-2 is predicted to remain prevalent for the foreseeable future, making the ability to identify SARS-CoV-2 variants imperative in understanding and controlling the pandemic. The predominant variant discovery method, genome sequencing, is time-consuming, insensitive, and expensive. Ultraperformance liquid chromatography–mass spectrometry (UPLC–MS) offers an exciting alternative detection modality provided that variant-containing peptide markers are sufficiently detectable from their tandem mass spectra (MS/MS). We have synthesized model tryptic peptides of SARS-CoV-2 variants alpha, beta, gamma, delta, and omicron and evaluated their signal intensity, HCD spectra, and reverse phase retention time. Detection limits of 781, 781, 65, and 65 amol are obtained for the molecular ions of the proteotypic peptides, beta (QIAPGQTGNIADYNYK), gamma (TQLPSAYTNSFTR), delta (VGGNYNYR), and omicron (TLVKQLSSK), from neat solutions. These detection limits are on par with the detection limits of a previously reported proteotypic peptide from the SARS-CoV-2 spike protein, HTPINLVR. This study demonstrates the potential to differentiate SARS-CoV-2 variants through their proteotypic peptides with an approach that is broadly applicable across a wide range of pathogens.

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Specific and Rapid SARS-CoV-2 Identification Based on LC-MS/MS Analysis

Cited by 2 publications

References 15 publications

Identification of SARS-CoV-2 Proteins from Nasopharyngeal Swabs Probed by Multiple Reaction Monitoring Tandem Mass Spectrometry

Identification of SARS-CoV-2 Proteins from Nasopharyngeal Swabs Probed by Multiple Reaction Monitoring Tandem Mass Spectrometry

Evaluation of Variant-Specific Peptides for Detection of SARS-CoV-2 Variants of Concern

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