Specific and Rapid SARS-CoV-2 Identification Based on LC-MS/MS Analysis

Abstract

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, emerged as the cause of a global crisis. Rapid and reliable clinical diagnosis is essential for effectively controlling transmission. The gold standard assay for SARS-CoV-2 identification is the highly sensitive real-time quantitative polymerase chain reaction (RT-qPCR); however, this assay depends on specialized reagents and may suffer from false results. Thus, additional assays based on different approaches could be beneficial. Here, we present a novel method for SARS-CoV-2 identification based on mass spectrometry. The approach we implemented combines a multistep procedure for the rational down-selection of a set of reliable markers out of all optional in silico derived tryptic peptides in viral proteins, followed by monitoring of peptides derived from tryptic digests of purified proteins, cell-cultured SARS-CoV-2, and nasopharyngeal (NP) swab matrix spiked with the virus. The marker selection was based on specificity to SARS-CoV-2 and on analytical parameters including sensitivity, linearity, and reproducibility. The final assay is based on six unique and specific peptide markers for SARS-CoV-2 identification. The simple and rapid (2.5 h) protocol we developed consists of virus heat inactivation and denaturation, tryptic digestion, and identification of the selected markers by liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/MS). The developed assay enabled the identification of 10⁴ PFU/mL SARS-CoV-2 spiked into buffer. Finally, the assay was successfully applied to 16 clinical samples diagnosed by RT-qPCR, achieving 94% concordance with the current gold standard assay. To conclude, the novel MS-based assay described here is specific, rapid, simple, and is believed to provide a complementary assay to the RT-qPCR method.

Figure 1

Methodology flowchart for SARS-CoV-2 marker selection. The first part was performed computationally, including in silico tryptic digest followed by length restriction (6–14 aa) and specificity consideration (removal of peptides that appear in human pathogen databases). The second part is LC-MS analysis of tryptic peptides derived from purified proteins, cell-cultured SARS-CoV-2, negative NP sample spiked with SARS-CoV-2, and positive NP samples. Evaluating parameters as sensitivity, reproducibility, and linearity helped reduce the number of potential markers.

Figure 2

Example of tryptic digest-derived marker behavior (AYNVTQAFGR, N protein, marker no. 3). (A) Extracted-ion chromatogram (EIC) of m/z 563.7856 from a full-scan LC-MS run of 10 ppm N protein (left). Linearity of a tryptic peptide derived from tryptic digestion of N protein in the range of 10 ppb–10 ppm (right). (B) Extracted-ion chromatogram (EIC) of m/z 563.7856 from a full-scan LC-MS run of 10⁶ PFU/mL SARS-CoV-2 (left). Linearity of a tryptic peptide derived from tryptic digestion of SARS-CoV-2 in the range of 10⁴–10⁶ PFU/mL (right). (C) Extracted-ion chromatogram (EIC) of m/z 563.7856 from a full-scan LC-MS run of 10⁶ SARS-CoV-2 after precipitation (left). Linearity of a tryptic peptide derived from tryptic digestion of SARS-CoV-2 in the range of 10³–10⁶ PFU/mL.

Figure 3

SARS-CoV-2 spiked to buffer or to NP-negative samples. SARS-CoV-2 was spiked to 50 mM ammonium bicarbonate buffer and to NP samples as is or diluted with 50 mM ammonium bicarbonate (2:3 respectively) to give 10⁶ PFU/mL. A reduction in peak intensities was observed in nondiluted matrix, while similar peak intensities in buffer and diluted matrix were observed monitoring our six selected markers.

Figure 4

LC-MS/MS (Orbitrap) identification of SARS-CoV-2-specific marker (FQTLLALHR, S protein, peptide no. 13). (A) Extracted-ion chromatogram (EIC) of m/z 366.8854 from a full-scan LC-MS run of 10⁶ PFU/mL SARS-CoV-2 spiked to buffer. (B) Extracted-ion chromatogram (EIC) of m/z 366.8854 from a full-scan LC-MS run of 10⁶ PFU/mL SARS-CoV-2 spiked to negative NP swab. (C) Extracted-ion chromatogram (EIC) of m/z 366.8854 from a full-scan LC-MS run of positive NP swab. (D) Extracted-ion chromatogram (EIC) of m/z 366.8854 from a full-scan LC-MS run of negative NP swab. (E) Mass spectrum of the specific marker FQTLLALHR (parent ion, (M + 3H)/3, at m/z 366.8856, chromatographic peak at 3.5 min) derived from spiked protein. (F) Marker fragmentation spectrum (MS-MS of 366.8854). Y—Ions that result from the cleavage of the C–N bonds (amide bond) of a peptide backbone with the C-terminal fragments retaining the charge. b—Ions that result from the cleavage of the C–N (amide bond) bonds of a peptide backbone with the N-terminal fragments retaining the charge. Z—Ions that result from the cleavage of the N–C bonds of a peptide backbone with the C-terminal fragments retaining the charge. i—Immonium ion is an internal fragment formed by a combination of a type (C–C bond) and y type (C–N bond) cleavage.