Identification of SARS-CoV‑2 Proteins from Nasopharyngeal Swabs Probed by Multiple Reaction Monitoring Tandem Mass Spectrometry

Numerous reverse
transcription polymerase chain reaction (RT-PCR)
tests have emerged over the past year as the gold standard for detecting
millions of cases of SARS-CoV-2 reported daily worldwide. However,
problems with critical shortages of key reagents such as PCR primers
and RNA extraction kits and unpredictable test reliability related
to high viral replication cycles have triggered the need for alternative
methodologies to PCR to detect specific COVID-19 proteins. Several
authors have developed methods based on liquid chromatography with
tandem mass spectrometry (LC–MS/MS) to confirm the potential
of the technique to detect two major proteins, the spike and the nucleoprotein,
of COVID-19. In the present work, an S-Trap mini spin column digestion
protocol was used for sample preparation prodromal to LC–MS/MS
analysis in multiple reactions monitoring ion mode (MRM) to obtain
a comprehensive method capable of detecting different viral proteins.
The developed method was applied to n. 81 oro/nasopharyngeal swabs
submitted in parallel to quantitative reverse transcription PCR (RT-qPCR)
assays to detect RdRP, the S and N genes specific for COVID-19, and
the E gene for all Sarbecoviruses, including SARS-CoV-2
(with cycle negativity threshold set to 40). A total of 23 peptides
representative of the six specific viral proteins were detected in
the monitoring of 128 transitions found to have good ionic currents
extracted in clinical samples that reacted differently to the PCR
assay. The best instrumental response came from the FLPFQFGR sequence
of spike [558−566] peptide used to test the analytical performance
of the method that has good sensitivity with a low false-negative
rate. Transition monitoring using a targeted MS approach has the great
potential to detect the fragmentation reactions of any peptide molecularly
defined by a specific amino acid sequence, offering the extensibility
of the approach to any viral sequence including derived variants and
thus providing insights into the development of new types of clinical
diagnostics.