Simultaneous monitoring of eight human respiratory viruses including SARS-CoV-2 using liquid chromatography-tandem mass spectrometry

Abstract

Diagnosis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection has primarily been achieved using reverse transcriptase polymerase chain reaction (RT-PCR) for acute infection, and serology for prior infection. Assay with RT-PCR provides data on presence or absence of viral RNA, with no information on virus replication competence, infectivity, or virus characterisation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is typically not used in clinical virology, despite its potential to provide supplemental data about the presence of viral proteins and thus the potential for replication-competent, transmissible virus. Using the SARS-CoV-2 as a model virus, we developed a fast 'bottom-up' proteomics workflow for discovery of target virus peptides using 'serum-free' culture conditions, providing high coverage of viral proteins without the need for protein or peptide fractionation techniques. This workflow was then applied to Coronaviruses OC43 and 229E, Influenza A/H1N1 and H3N2, Influenza B, and Respiratory Syncytial Viruses A and B. Finally, we created an LC-MS/MS method for targeted detection of the eight-virus panel in clinical specimens, successfully detecting peptides from the SARS-CoV-2 ORF9B and nucleoprotein in RT-PCR positive samples. The method provides specific detection of respiratory viruses from clinical samples containing moderate viral loads and is an important further step to the use of LC-MS/MS in diagnosis of viral infection.

Figure 1

(Left) Box-and-whisker plots of % peak area (TIC-normalised total SWATH fragment peak area) for all detected SARS-CoV-2 peptides in culture digests, showing improved detectability of virus proteins despite overall lower sample loading. (Middle) Overlay of total ion chromatograms (TIC) for SARS-CoV-2 culture digests under standard culturing conditions (2% FBS) and serum-free conditions. Overall higher intensity of signal for FBS culture represents higher total protein loading on the LC–MS. (Right) Box-and-whisker comparison of normalised peak areas for all targeted peptides in spiked samples. The area of each peak in the MRM chromatogram was normalised to the total area under the chromatogram within the detection window.

Figure 2

(Left) Heat map of signal-to-noise ratio for combined peak areas of virus spikes (1:9 v/v) versus peak area average of blank samples. (Right) Overlay of MRM chromatograms from human background proteins (upper panel) and virus cultures (lower panel) showing efficient use of MS detection time and minimal concurrency through careful peptide selection and maximising chromatographic separation.

Figure 3

Graphical summary of workflow from viral cultures to rapid targeted MRM method. Use of serum-free culture avoids the need for extensive sample fractionation and use of both data-dependent and data-independent-acquisition methods during discovery phase streamlines peptide selection process. Created with BioRender.com.

Figure 4

Targeted method performance for six peptides targeting 3 proteins from SARS-CoV-2. (Top panel) Spread of Ct values for 3 SARS-CoV-2 genes vs # peptides detected in clinical sample cohort 3. (Lower panel) From left; (A) Violin plot comparing software generated signal-to-noise ratio between positive (left) and negative (right) clinical specimens. (B) Correlation between reported N-gene Ct and peak area of the most intense MRM transition. Grey dots are peak areas detected in RT-PCR negative samples, assigned an arbitrary Ct of 25 for ease of reading. (C) linear regression of best two MRMs for each peptide for all RT-PCR positive samples. (D, top panel) linear regression and correlation for raw peak area and analyte/IS peak area ratio for peptide N.AYNTQAEFGR, which has been reported in other published targeted methods. (D, lower three panels) linear regression and correlation of other SARS-CoV-2 peptide peak areas to N.AYNTQAEFGR.

Figure 5

Targeted method imprecision (repeated preparations of pooled sample, n = 5) and repeatability (repeated injections of pooled sample extract, n = 6). Results are combined for 6 SARS-Cov-2 peptides using CVs for peak area, IS peak area and peak area ratio, and for 6 background peptides for peak area only.