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INTRODUCTIONThe pandemic readiness toolbox needs to be extended, providing diagnostic tools that target different biomolecules, using orthogonal experimental setups and fit-for-purpose specification of detection. Here we build on a previous Cov-MS effort that used liquid chromatography-mass spectrometry (LC-MS) and describe a method that allows accurate, high throughput measurement of SARS-CoV-2 nucleocapsid (N) protein. MATERIALS and METHODSWe used Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) technology to enrich and quantify proteotypic peptides of the N protein from trypsin-digested samples from COVID-19 patients. RESULTSThe Cov2MS assay was shown to be compatible with a variety of sample matrices including nasopharyngeal swabs, saliva and blood plasma and increased the sensitivity into the attomole range, up to a 1000-fold increase compared to direct detection in matrix. In addition, a strong positive correlation was observed between the SISCAPA antigen assay and qPCR detection beyond a quantification cycle (Cq) of 30-31, the level where no live virus can be cultured from patients. The automatable "addition only" sample preparation, digestion protocol, peptide enrichment and subsequent reduced dependency upon LC allow analysis of up to 500 samples per day per MS instrument. Importantly, peptide enrichment allowed detection of N protein in a pooled sample containing a single PCR positive sample mixed with 31 PCR negative samples, without loss in sensitivity. MS can easily be multiplexed and we also propose target peptides for Influenza A and B virus detection. CONCLUSIONSThe Cov2MS assay described is agnostic with respect to the sample matrix or pooling strategy used for increasing throughput and can be easily multiplexed. Additionally, the assay eliminates interferences due to protein-protein interactions including those caused by anti-virus antibodies. The assay can be adapted to test for many different pathogens and could provide a tool enabling longitudinal epidemiological monitoring of large numbers of pathogens within a population, applied as an early warning system.
RESUMO
Reliable, robust, large-scale molecular testing for SARS-CoV-2 is essential for monitoring the ongoing Covid-19 pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immunoaffinity enrichment combined with liquid chromatography - mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in PBS swab media from combined throat/nasopharynx/saliva samples. The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their corresponding RT-PCR readout (r=0.79). The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with a quantitative readout of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 21 to 34. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% negative percent agreement (estimated specificity) and 95% positive percent agreement (estimated sensitivity) when analyzing clinical samples collected from asymptomatic individuals with a Ct within the limit of detection of the mass spectrometer (Ct [≤]30). These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies.
