Targeted proteomics as a tool to detect SARS-CoV-2 proteins in clinical specimens

Abstract

The rapid, sensitive and specific detection of SARS-CoV-2 is critical in responding to the current COVID-19 outbreak. In this proof-of-concept study, we explored the potential of targeted mass spectrometry (MS) based proteomics for the detection of SARS-CoV-2 proteins in both research samples and clinical specimens. First, we assessed the limit of detection for several SARS-CoV-2 proteins by parallel reaction monitoring (PRM) MS in infected Vero E6 cells. For tryptic peptides of Nucleocapsid protein, the limit of detection was estimated to be in the mid-attomole range (9E-13 g). Next, this PRM methodology was applied to the detection of viral proteins in various COVID-19 patient clinical specimens, such as sputum and nasopharyngeal swabs. SARS-CoV-2 proteins were detected in these samples with high sensitivity in all specimens with PCR Ct values <24 and in several samples with higher CT values. A clear relationship was observed between summed MS peak intensities for SARS-CoV-2 proteins and Ct values reflecting the abundance of viral RNA. Taken together, these results suggest that targeted MS based proteomics may have the potential to be used as an additional tool in COVID-19 diagnostics.

Fig 1. Numbers of identified proteins in SARS-CoV-2 infected Vero cells (PD2.3/Mascot search engine, offline high pH RP fractionation into four fractions, total input material 0.6 μg, 90 min LC gradients on an Orbitrap Lumos).

Fig 2. PRM results visualized in skyline (skyline.ms).

Chromatograms for each of the Top6 fragment ions are shown in different colors in a dilution series for tryptic peptides A) GFYAEGSR (NCAP_SARS2), B) ADETQALPQR (NCAP_SARS2) and C) EITVATSR (VME1_SARS2). The lower right chromatogram represents the lowest sample input, i.e. 20 pg. The MS/MS spectrum on the right is the library spectrum. C) Calibration curves based on PRM data for three target peptides recorded on an Orbitrap Eclipse. The summed AUC values for the Top6 fragment ions of each peptide were taken for relative quantitation. ‘Input’ is total protein input from the SARS2-CoV-2 infected Vero E6 cell lysate; inserts are zoom-ins of the input range 0–300 pg.

Fig 3. PRM fragment ion chromatograms of SARS-CoV-2 Nucleocapsid and VME1 tryptic peptides VAGDSFAAYSR and AYNVTQAFGR in representative A) sputum specimens and B) throat swab specimens of COVID-19 patients.

Chromatograms for each of the Top6 fragment ions are shown in different colors. The upper panels show the fragment ion chromatograms of the corresponding synthetic AQUA peptides VAGDSFAAYS[R] (m/z 605.79) and AYNVTQAFG[R] (m/z 568.79). See S4 Fig for additional clinical specimens.

Fig 4. PRM data of clinical specimens of COVID-19 patients (cohort 1).

A) Total AUCs of SARS-CoV-2 target peptide fragment ion chromatograms (upper panels show the spiked-in AQUA peptide signals; if no AQUA peptide counterpart was available upper panels are left empty). The color shading of the bars indicate the relative AUCs of the different fragment ions. B) Fragment ion chromatograms for various SARS-CoV-2 target peptides in one representative clinical specimen. C) Comparison of AUCs versus PCR Ct values for clinical specimens. Data points in grey represent samples in which no target peptides were detected by PRM.

Fig 5. PRM data of clinical specimens of COVID-19 patients (cohort 2).

A) Total AUCs of SARS-CoV-2 target peptide fragment ion chromatograms (upper panels show the spiked-in AQUA peptide signals; if no AQUA peptide counterpart was available upper panels are left empty). The color shading of the bars indicate the relative AUCs of the different fragment ions. B) Selection of PRM results for several target peptides in samples 2, 4 and 7. See main text for explanation. Retention times for the identical peptides in different samples may slightly differ as a result of small variations in LC gradients and chromatography setup. C) Comparison of AUCs versus PCR Ct values for clinical specimens. Data points in grey represent samples in which no target peptides were detected by PRM.

Fig 6

A) Comparison of one-shot versus high pH fractionation LC-MS PRM for several target peptides. For peptide GFYQTSNFR in Sample #7 the normalized peak area would correspond to the low attomolar range. B) Example of a positive target peptide identification in a 20 min gradient LC-MS run of a sample of high Ct value.