Targeted proteomics for detection of SARS-CoV-2 proteins in clinical samples

By Colin Lightfoot, M.Sc. Infection and ImmunityNov 16 2021

The current global coronavirus 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The COVID pandemic has been described by the World Health Organization (WHO) as a public health emergency of international concern.

To date, there have been more than five million deaths reported due to COVID-19, and this number is believed to be an underestimation due to the lack of testing capacity in developing parts of the world.

Other than the more conventional polymerase chain reaction (PCR) approach, immunoassays have been utilized to detect viruses. Additionally, mass spectrometry (MS) based techniques have been employed for the detection of influenza viral proteins and human metapneumovirus (HMPV) in clinical samples. A substantial increase has been observed from recent developments in targeted proteomics methods and Orbitrap mass spectrometry, such as parallel reaction monitoring (PRM). Several SARS-CoV-2 studies have utilized MS approaches, but it is not yet clear if state-of-the-art proteomic technologies could provide the sensitivity and specificity needed for definitive diagnoses.

A team of researchers from various institutions within the Netherlands examined the use of targeted MS-based proteomics for the detection of SARS-CoV-2 in research and clinical samples. The authors first analyzed the limit of detection of PRM on an Orbitrap mass spectrometer for specific tryptic peptides of SARS-CoV-2 proteins. The authors then tested the sensitivity of this method for the detection of SARS-CoV-2 in clinical specimens, such as nasopharyngeal swabs, sputum, and mucus.

This study is published in 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 study

The authors used standard bottom-up proteomics to analyze the global proteome of Vere E6 cells that were infected with SARS-CoV-2. This targeted proteomics method was then employed to detect SARS-CoV-2 proteins in samples from COVID-19 patients. Due to all viral infectivity in the specimens needing to be abolished in a biosafety level three (BSL-3) laboratory before any further processing occurs, the condition of the starting material was not optimized for subsequent proteomics.

The PRM assay for the first patient cohort had relatively high mass spectral peak intensities for the various target peptides in all specimens. Also, viral proteins could still be unambiguously detected even in several specimens with cycle threshold (Ct) values in the low twenties. For example, one of the sample peptides (#5 peptide) GQGVPINTNSSPDDQIGYYR was identified by eight highly mass accurate fragment ions. Between the sample characteristics, there is a clear inverse relationship, with a threshold value for detection via targeted mass spectrometry being around a Ct value of 22.

The second patient sample cohort was comprised of fifteen nasopharyngeal swabs collected from patients who were COVID-19 positive. Again, the authors conducted positive MS, which revealed all patients with Ct values greater than 24, even though the absolute summed intensities of target peptide fragments displayed wide variation. Surprisingly, different sets of target peptides were more strongly detected in several patient samples, even though each sample was prepared exactly the same way, following the same protocol. This could be due to sample heterogeneity, which may have caused diverse outcomes of protein digestion.

To obtain higher sensitivity and decrease the overall liquid chromatography MS analysis time, the authors tested two different experimental procedures. First, they applied high pH reversed-phase fractionation to tryptic digests of clinical samples to increase the measurement of sensitivity. Second, fractionated peptides were collected in eight fractions, which were analyzed separately by PRM MS. This procedure resulted in some cases to improved detection of peptides and higher quantification values.

In the fractional samples, peptide abundances were up to five times higher, while absolute quantitation based on comparison to estimated spiked in amounts of AQUA counterpart peptides showed that SARS-CoV-2 peptides could be detected in the low- to the mid-attomolar range. There were reduced identifications and quantitation results with a shorter liquid chromatography MS gradient. Although, extremely low levels of target peptides could still be identified and quantified, even with the increased presence of contaminating peaks that were most likely due to more crowded mass spectra.

Implications

The current sensitivity levels of PRM proteomics methodology and the successful identification of SARS-CoV-2 proteins in clinical samples allow for the exploration of mass spectrometry as a method for clinical and diagnostics laboratories to detect SARS-CoV-2 infections in clinical specimens.

Future research into these methods should focus on the optimization of fast sample preparation procedures and liquid chromatography-mass spectrometry throughput.