The on-going SARS-CoV-2 (COVID-19) pandemic has called for an urgent need for rapid and high-throughput methods for mass testing for early detection, prevention and surveillance of the disease. We investigated whether targeted parallel reaction monitoring (PRM) quantification using high resolution Orbitrap instruments can provide the sensitivity and speed required for a high-throughput method that could be used for clinical diagnosis. We developed a high-throughput and sensitive PRM-MS assay that enables absolute quantification of SARS-CoV-2 nucleocapsid peptides with short turn-around times by using isotopically labelled synthetic SARS-CoV-2 concatenated peptides. We established a fast and high-throughput S-trap-based sample preparation method, which was then utilized for testing 25 positive and 25 negative heat-inactivated nasopharyngeal swab samples for SARS-CoV-2 detection. The method was able to differentiate between negative and some of the positive patients with high viral load .Moreover, based on the absolute quantification calculations, our data show that patients with Ct values as low as 17.5 correspond to NCAP protein amounts of around 7.5 pmol in swab samples. The present high-throughput method could potentially be utilized in specialized clinics as an alternative tool for detection of SARS-CoV-2 but will require enrichment of viral proteins in order to compete with RT-qPCR.

A targeted PRM method for detection of diagnostic peptides originating from SARS-CoV-2 NCAP protein in 50 (25 positive and 25 negative) clinical patient samples. SIL peptides (CoV-MS QconCAT) was spiked in before digestion for peptide verification as well as absolute quantification.

Clinical swab samples from COVID-19 positive and negative patients were collected in various NHS testing facilities and shipped in viral transfer media (VTM).