Cov-MS: a community-based template assay for clinical MS-based protein detection in SARS-CoV-2 patients

Abstract

Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by Reverse Transcription Polymerase Chain Reaction (RT-PCR). Although this is efficient, automatable and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using Mass Spectrometry (MS). We established the Cov-MS consortium, consisting of fifteen academic labs and several industrial partners to increase applicability, accessibility, sensitivity and robustness of this kind of SARS-CoV-2 detection. This in turn gave rise to the Cov-MS Digital Incubator that allows other labs to join the effort, navigate and share their optimizations, and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR, using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550.

A Rapid and Reliable Liquid Chromatography/Mass Spectrometry Method for SARS-CoV-2 Diagnostics from Gargle Solutions and Saliva

“…It has been demonstrated that 10 genomes, i.e. DNA plasmids, 11 can be detected in a volume of 10 µl with cycle times (ct) of 38 in RT-qPCR experiments. A RT-qPCR detection with a ct value of 18 would require the presence of 10,485,760 genome equivalents in 10 µl.…”

“…Calculations with 300 molecules of nucleoprotein per SARS-CoV-2 virus particle 11 result in a nucleoprotein concentration of ~ 520 amol/µl, corresponding to a ct value of 18. 11 In our experiments, we used 100 µl of saliva and generated 70 µl of sample solution for LC/MS analysis. Under these conditions, the concentration of SARS-CoV-2 nucleoprotein peptides would be 743 amol/µl, which is above our estimated LOD/LOQ of ~ 500 amol/µl (see above).…”

“…The copyright holder for this preprint this version posted May 7, 2021. ; https://doi.org/10.1101/2021.05.05.21256257 doi: medRxiv preprint It is however very likely that a "real-world" RT-qPCR experiment is less sensitive than the one performed under the ideal conditions described in 11 as RNA recovery is rarely complete. Therefore, RT-qPCR experiments with specific ct values would be based on higher virus numbers, resulting in de facto higher nucleoprotein concentrations.…”

“…5 In the meantime, the potential of MS has become evident 6 and numerous reports on SARS-CoV-2 identification by MS methods have been published since the beginning of the pandemic. [7][8][9][10][11] In the present work, we describe our latest efforts in developing a rapid liquid chromatography (LC)/MS method for SARS-CoV-2 diagnostics from gargle solutions and saliva. We report an…”

“…5 In the meantime, the potential of MS has become evident 6 and numerous reports on SARS-CoV-2 identification by MS methods have been published since the beginning of the pandemic. [7][8][9][10][11] In the present work, we describe our latest efforts in developing a rapid liquid chromatography (LC)/MS method for SARS-CoV-2 diagnostics from gargle solutions and saliva. We report an MS-compatible sample preparation strategy and a quantification protocol that is based on selected peptides from SARS-CoV-2 nucleoprotein by an LC/multiple-reaction monitoring (MRM)-MS approach.…”