A fast and sensitive absolute quantification assay for the detection of SARS-CoV-2 peptides using parallel reaction...

The on-going SARS-CoV-2 (COVID-19) pandemic has called for an urgent need for rapid and high-throughput methods for mass testing and early detection, prevention as well as 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 and utilized it for testing 25 positive and 25 negative heat-inactivated clinical 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 17.8 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.