Cov²MS: an automated matrix-independent assay for mass spectrometric detection and measurement of SARS-CoV-2 nucleocapsid protein

ABSTRACT: Introduction. The 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 to use liquid chromatography coupled to mass spectrometry (LC-MS) and describe a method developed to allow accurate, high throughput measurement of SARS-CoV-2 nucleocapsid (NCAP) protein. Methods. We use 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. Results. This makes the Cov²MS assay compatible with most matrices including nasopharyngeal swabs, saliva and blood plasma, while increasing the sensitivity into the attomole range, up to a 1000-fold increase compared to direct detection in matrix. In addition, there is a strong positive correlation between the SISCAPA antigen assay and qPCR detection up to a quantification cycle (Cq) of 30. The automatable “addition only” sample preparation and digestion protocol, the peptide enrichment and the reduced dependency upon LC together allow analysis of up to 500 samples per day per MS instrument. 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. We also propose novel target peptides for Influenza A and B. Conclusion. Since the Cov²MS assay is insensitive to matrix or pooling and easily multiplexed, it can be used to test for many different pathogens and could provide longitudinal epidemiological monitoring of large numbers of pathogens within a population, applied as an early warning system.

ORGANISM(S): Severe Acute Respiratory Syndrome Coronavirus 2

SUBMITTER: Bart Van Puyvelde  

PROVIDER: PXD031401 | panorama | Mon Feb 17 00:00:00 GMT 2025

REPOSITORIES: PanoramaPublic