Focused proteomics for detection of SARS-CoV-2 proteins in medical samples

The present world coronavirus 2019 (COVID-19) pandemic is attributable to extreme acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The COVID pandemic has been described by the World Well being Group (WHO) as a public well being emergency of worldwide concern.

So far, there have been greater than 5 million deaths reported as a consequence of COVID-19, and this quantity is believed to be an underestimation because of the lack of testing capability in growing components of the world.

Aside from the extra standard polymerase chain response (PCR) method, immunoassays have been utilized to detect viruses. Moreover, mass spectrometry (MS) based mostly methods have been employed for the detection of influenza viral proteins and human metapneumovirus (HMPV) in medical samples. A considerable enhance has been noticed from current developments in focused proteomics strategies and Orbitrap mass spectrometry, corresponding to parallel response monitoring (PRM). A number of SARS-CoV-2 research have utilized MS approaches, however it’s not but clear if state-of-the-art proteomic applied sciences might present the sensitivity and specificity wanted for definitive diagnoses.

A group of researchers from varied establishments inside the Netherlands examined using focused MS-based proteomics for the detection of SARS-CoV-2 in analysis and medical samples. The authors first analyzed the restrict of detection of PRM on an Orbitrap mass spectrometer for particular tryptic peptides of SARS-CoV-2 proteins. The authors then examined the sensitivity of this technique for the detection of SARS-CoV-2 in medical specimens, corresponding to nasopharyngeal swabs, sputum, and mucus.

This examine is revealed within the PLOS One journal.

Study: Targeted proteomics as a tool to detect SARS-CoV-2 proteins in clinical specimens. Image Credit: Pong Ch / Shutterstock

The examine

The authors used commonplace bottom-up proteomics to investigate the worldwide proteome of Vere E6 cells that had been contaminated with SARS-CoV-2. This focused proteomics technique was then employed to detect SARS-CoV-2 proteins in samples from COVID-19 sufferers. On account of all viral infectivity within the specimens needing to be abolished in a biosafety stage three (BSL-3) laboratory earlier than any additional processing happens, the situation of the beginning materials was not optimized for subsequent proteomics.

The PRM assay for the primary affected person cohort had comparatively excessive mass spectral peak intensities for the varied goal peptides in all specimens. Additionally, viral proteins might nonetheless be unambiguously detected even in a number of specimens with cycle threshold (Ct) values within the low twenties. For instance, one of many pattern peptides (#5 peptide) GQGVPINTNSSPDDQIGYYR was recognized by eight extremely mass correct fragment ions. Between the pattern traits, there’s a clear inverse relationship, with a threshold worth for detection by way of focused mass spectrometry being round a Ct worth of twenty-two.

The second affected person pattern cohort was comprised of fifteen nasopharyngeal swabs collected from sufferers who had been COVID-19 constructive. Once more, the authors carried out constructive MS, which revealed all sufferers with Ct values better than 24, regardless that absolutely the summed intensities of goal peptide fragments displayed extensive variation. Surprisingly, totally different units of goal peptides had been extra strongly detected in a number of affected person samples, regardless that every pattern was ready precisely the identical manner, following the identical protocol. This may very well be as a consequence of pattern heterogeneity, which can have induced various outcomes of protein digestion.

To acquire greater sensitivity and reduce the general liquid chromatography MS evaluation time, the authors examined two totally different experimental procedures. First, they utilized excessive pH reversed-phase fractionation to tryptic digests of medical samples to extend the measurement of sensitivity. Second, fractionated peptides had been collected in eight fractions, which had been analyzed individually by PRM MS. This process resulted in some circumstances to improved detection of peptides and better quantification values.

Within the fractional samples, peptide abundances had been as much as 5 occasions greater, whereas absolute quantitation based mostly on comparability to estimated spiked in quantities of AQUA counterpart peptides confirmed that SARS-CoV-2 peptides may very well be detected within the low- to the mid-attomolar vary. There have been diminished identifications and quantitation outcomes with a shorter liquid chromatography MS gradient. Though, extraordinarily low ranges of goal peptides might nonetheless be recognized and quantified, even with the elevated presence of contaminating peaks that had been probably as a consequence of extra crowded mass spectra.


The present sensitivity ranges of PRM proteomics methodology and the profitable identification of SARS-CoV-2 proteins in medical samples enable for the exploration of mass spectrometry as a way for medical and diagnostics laboratories to detect SARS-CoV-2 infections in medical specimens.

Future analysis into these strategies ought to give attention to the optimization of quick pattern preparation procedures and liquid chromatography-mass spectrometry throughput.

Journal reference:

  • Focused proteomics as a software to detect SARS-CoV-2 proteins in medical specimens, Karel Bezstarosti,Mart M. Lamers,Wouter A. S. Doff,Peter C. Wever,Khoa T. D. Thai,Jeroen J. A. van Kampen,Bart L. Haagmans,Jeroen A. A. Demmer, PLOS ONE, 2021.11.11.

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