Rapid and sensitive detection of SARS-CoV-2 infection using quantitative peptide enrichment LC-MS analysis

Abstract

Reliable, robust, large-scale molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for monitoring the ongoing coronavirus disease 2019 (COVID-19) pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immuno-affinity enrichment combined with liquid chromatography-mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in phosphate-buffered saline (PBS) swab media from combined throat/nasopharynx/saliva samples. The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their correspondingreal-time polymerase chain reaction (RT-PCR) read-out (r = 0.79). The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with a quantitative read-out of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 21 to 34. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% negative percent agreement (estimated specificity) and 95% positive percent agreement (estimated sensitivity) when analyzing clinical samples collected from asymptomatic individuals with a Ct within the limit of detection of the mass spectrometer (Ct ≤ 30). These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies.

Cov2MS: an automated matrix-independent assay for mass spectrometric detection and measurement of SARS-CoV-2 nucleocapsid protein in infectious patients

“…As Cov 2 MS can now measure positive patients up to Cq 30 in UTM, this implies close to a 1000-fold increase in assay sensitivity for patient samples in UTM matrix. Importantly, this patient data also very strongly resembles the results obtained by Hober et al, showing effective inter-laboratory roll-out 14 .…”

“…LogInt of the AYN peptide (green bars, left axis) and the Cq value for the E-gene (red dots, right axis). As reported earlier, the LogInt of positive patients strongly correlates linearly (R 2 = 0.86) to the Cq value, which is an exponential metric depicting the number of doublings required for detection 2,14 . In both plots, patients were sorted from low to high virus measurement, i.e.…”

“…As suggested by Hober et al, we also plotted these numbers against a qPCR positivity threshold of Cq 30 - leaving out the patients between Cq 30 and 40. This clearly illustrates how well qPCR and MS agree, especially up to Cq 30, with 96.2% PPA and 98.2% NPA, respectively 14 .…”

“Multiomics” Approaches to Understand and Treat COVID-19: Mass Spectrometry and Next-Generation Sequencing

“…This method has been able to discern the SARS-CoV-2 nucleoprotein NCAP_SARS2 from 1500 distinguished proteins in COVID-19 positive nasopharyngeal samples (which were already determined as positive through quantitative reverse transcription PCR) [54]. Compared to methods such as RT-PCR and rapid tests, LC-MS/MS has shown both high sensitivity and specificity while also decreasing the risk of false-positives [41,55]. These findings support the use of MS-based proteomics in the diagnosis of COVID-19.…”

Add mass spectrometry to the pandemic toolbox

“…In response, scientists usually strive to improve the signal-to-noise ratio by increasing the signal. Now, in eLife, Fredrik Edfors and colleagues at institutions in Sweden and Canada – including Andreas Hober as first author – report having tweaked how to prepare a mass spectrometry sample to prevent background noise from emerging instead ( Hober et al, 2021 ).…”