Cov-MS: a community-based template assay for clinical MS-based protein detection in Sars-Cov-2 patients
In preparation (preprint on medRxiv)
- Organism: SARS-CoV-2, Homo sapiens
- Instrument: Xevo TQ-S,Xevo TQ-XS,TripleTOF 6600,TSQ Altis,Triple Quad 5500,6495A Triple Quadrupole LC/MS,Triple Quad 6500,Xevo TQD
SARS-Cov-2, proteomics, mass spectrometry, nasopharyngeal swabs, multiple reaction monitoring, QconCAT
Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes the coronavirus disease (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 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.
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A SARS-CoV-2 spectral library was generated from a dilution series of two recombinant proteins (NCAP_SARS2 and SPIKE_SARS2) in UTM medium using narrow- and wide-window DIA data (SWATH). The library was applied on 20 blind patient samples with Ct-values ranging from 12-20. 17 responsive peptides from both NCAP_SARS2 and SPIKE_SARS2 were retained and optimized for a Multiple Reaction Monitoring (MRM) assay on a Waters Xevo TQ-S. Other labs were invited to the Cov-MS consortium to validate the MRM method on other vendor platforms (SCIEX, Thermo and Agilent). Additionally, a QconCAT heavy standard containing the 17 target peptides, 4 histone peptides and 3 RepliCal peptides was kindly provided by Polyquant. Finally, a batch of 300 nasofaryngeal patient samples was analysed in MRM using a Waters Xevo TQ-XS.
50µl patient sample (1/60) was precipitated by adding 450µl (9 volumes) of ice-cold acetone (-20°C). After spinning at 16.000g and 0°C, the supernatant was discarded and 1µg of Trypsin/Lys-C mix (Promega) in 60µl 500 mM triethylammonium bicarbonate (TEABC) buffer was added. This was followed by an incubation step of four hours at 37°C, to facilitate trypsin digestion. Next, 20µl of this sample was prepared for analysis in a final concentration of 0.1% formic acid (FA) of which 2µl was injected into the LC-MS system. The protocol was validated on a dilution series of two recombinant Covid-19 proteins NCAP_SARS2 and SPIKE_SARS2 (Sino Biological, Beijing, China) which were found to be most abundant in public data on SARS-COV 2. A triplicate dilution series of 250 µL of negative patient UTM medium was spiked with different amounts of recombinant protein (500, 100, 50 ,10, 5, 1, 0.5, 0.1, 0.05, 0ng) resulting in a concentration range of 1ng-300fg on column when injecting 2 µL on column.
Created on 11/21/20, 5:26 PM