COVID-19 vaccines are continuing to become more widely available, but accurate and rapid testing remains a crucial tool for slowing the spread of the SARS-CoV-2 virus. Although quantitative reverse transcription-polymerase chain reaction (qRT-PCR) remains the most prevalent testing methodology, numerous tests have been developed that are predicated on detection of the SARS-CoV-2 nucleocapsid protein, including liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoassay based approaches. The continuing emergence of SARS-CoV-2 variants has complicated these approaches, as both qRT-PCR and antigen detection methods can be prone to missing viral variants. In this study, we describe a number of cases with COVID-19 where we were unable to detect the expected peptide targets from clinical nasopharyngeal swab samples that are typically identifiable in a targeted mass spectrometric assay. Whole genome sequencing revealed that single nucleotide polymorphisms in the gene encoding the viral nucleocapsid protein led to sequence variants that were not monitored in the targeted assay. Small modifications to the LC-MS/MS method ensured detection of the variants of the target peptide. Additional nucleocapsid variants were detected by performing bottom-up proteomic analysis of whole viral genome sequenced samples. This study demonstrates the importance of considering variants of SARS-CoV-2 in the assay design and highlights the flexibility of mass spectrometry-based approaches to detect variants as they evolve.

We recently completed a study to evaluate multiple testing platforms for analysis of 350 clinical nasopharyngeal swab samples including LC-MS/MS, point-of-care tests, qRT-PCR, and digital droplet PCR (manuscript submitted). The samples included 250 SARS-CoV-2 positive samples and 100 negative samples as determined by RT-PCR. A correlation of the concentration results for the AYNVTQAFGR and QQTVTLLPAADLDDFSK target peptides was performed which is found to be imperfect in part because the concentration of many of these samples had concentrations above the calibration curve (top calibrator=100 pM); but, the majority of samples produced the expected results of equimolar concentrations for the two target peptides. However, we observed 11 samples with a high concentration of the AYNVTQAFGR peptide and the abundance of QQTVTLLPAADLDDFSK being below the lower limit of quantitation (LLOQ). To investigate if the peptide was not identified because of a sequence variation, viral genome sequencing was performed for the 11 samples. Single nucleotide polymorphisms (SNPs) causing corresponding single amino acid substitutions that alter the sequence of the QQTVTLLPAADLDDFSK target peptide were confirmed by sequencing in 10/11 samples. We observed two variants in the peptide – QQTVTLLPAADLDDFSK-Q389L (LQTVTLLPAADLDDFSK) and T391I (QQIVTLLPAADLDDFSK), both of which resulted in an alteration of precursor ion mass, which explained our inability to detect the second target peptide.

All clinical samples were de-identified prior to analysis. Nasopharyngeal (NP) swab samples were collected in PBS and 750 μL of the sample was transferred to a 96 well plate. The virus was inactivated by adding 15 μL of Z3-16 and incubating at 70°C for 30 minutes. Following a 10 min cooling period at 4°C, antibody-based purification was performed using the anti-nucleocapsid protein monoclonal antibody coupled to MSIA D.A.R.T.’S. The purification procedure was conducted using the automated Versette liquid handling system. The tips were first washed with 1X PBS and then nucleocapsid protein was captured over a period of 1.75 hours. Following capture, the tips with were washed twice with 300 μL of 1X PBS and then 300 μL of water. The nucleocapsid protein was eluted with 100 μL of 0.2% TFA and 0.002% Z3-16 in water. The purified sample was immediately trypsin digested (rapid digest kit, Catalog#VA1060, Promega, Madison, WI).