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

Abstract

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.

Figure 1

Workflow of experimental strategy consisting of a preliminary trial phase crucial to develop the LC-MRM/MS method and a proposed assay to combine data from two molecular approaches.

Figure 2

Extraction of ion chromatogram (EIC) of peptides from the S1 portion (f16-685) of the SARS-COVID-2 spike. The most abundant, e.g., 558–566, was monitored in negative swabs with standard proteins at different concentrations. A comparison of the extracted ion chromatogram (EIC) between the outer and inner standards was reported in the lower panel.

Figure 3

MRM TIC chromatograms of four peptides (346–354, 444–453, 558–566, and 634–645) of the SARS-CoV-2 spike protein detected in qPCR-positive swab samples.