ABSTRACT
Background-aim: A lesson learned already in the early phase of the COVID-19 pandemic is the need for diagnostic tools that target different biomolecules, using orthogonal experimental setups and fit-for-purpose specification of detection, in addition to the well accepted reverse transcription polymerase chain reaction (RT-PCR). Methods: The Cov-MS effort developed an isotope dilution (based on QconCAT technology) - liquid chromatography mass spectrometry (LC-MS) method that allows accurate, high throughput measurement of SARS-CoV-2 nucleocapsid (NCAP) protein. It uses Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) technology to enrich and quantify proteotypic peptides of the NCAP protein from trypsin-digested samples from COVID-19 patients. The method is for a bigger part automatable (in terms of the sample preparation, digestion, peptide enrichment and LC-MS measurements). Results: The Cov2MS assay is compatible with most matrices including nasopharyngeal swabs, saliva and blood plasma, with a sensitivity into the attomole range thanks to the peptide enrichment. The latter also reduces dependency upon LC and allows shortening of LC run time, resulting in the analysis of up to 500 samples per day per MS instrument. There is a strong positive correlation between the SISCAPA antigen assay and qPCR detection up to a Cycle threshold (Ct) of 30. Importantly, peptide enrichment allowed detection of NCAP protein in a pooled sample containing a single PCR positive patient mixed with 31 PCR negative samples, without loss in sensitivity. Finally, we also demonstrated that it is feasible to rapidly adapt the method for the incorporation of ever emerging variants of concern (VoC), and even other types of respiratory viruses (e.g. Influenza A and B). Conclusions: In conclusion, since the Cov2MS assay is insensitive to pooling and easily multiplexed, it can provide longitudinal epidemiological monitoring of large numbers of pathogens within a population and can be applied as an early warning system.