Researchers investigate using mass spectrometry to detect viral proteins as a new method to test for the novel coronavirus
This web page was produced as an assignment for an undergraduate course at Davidson College.
In 2019 the world saw the first cases of SARS-CoV-2, which spiraled out into a pandemic. Throughout this unprecedented time, a key component of containing the disease has been our ability to rapidly test for and diagnose COVID-19, for which the real-time RT-PCR test is widely used to identify RNA from SARS-CoV-2 (Esbin et al. 2020). When talking about diagnostic tests such as those for COVID-19, the terms most often used are sensitivity and specificity. Sensitivity refers to how sensitive the test is to positive cases. The percentage of positive cases that the test says are positive is the test’s sensitivity. Specificity refers to the test’s ability to identify the specific disease, and not give a positive result unless they are infected. It asks how many negative cases resulted in a negative test. Real-time RT-PCR is the current gold standard for detecting the novel coronavirus (Esbin et al. 2020), and has approximately 95% sensitivity (confirmed by two different laboratories) and 100% specificity in the lab (Corman et al. 2020). While these numbers are hard to beat, Cardozo et al. wanted to create a new method of testing that would have high specificity, good sensitivity compared to PCR, and require the shortest amount of prep time while still allowing a larger number of samples to be tested at once. Unlike real-time RT-PCR, this method identifies coronavirus proteins using mass spectrometry (MS), a method which has been highly successful for other microbial identifications (Cardozo et al. 2020). Their hypothesis also involved developing a method for high-throughput testing with a short turnaround time, which would be an improvement as PCR has trouble identifying samples with a low viral load in pooled testing (“COVID-19 RT-PCR Test – EUA Summary” 2021 p. 19). If successful, this proteomic approach would help ease the demand for PCR test-related supplies and make it possible to do screening on a larger scale.
Cardozo et al. investigated peptides from SARS-CoV-2 that were correlated to a high viral load, and narrowed the list to nine nucleoprotein peptides unique to the virus, of which two were the most easily detectable (known in short form as DGI and IGM). To test for the virus, this method uses two processes in tandem — turbulent flow chromatography and mass spectrometry (TFC-MS/MS). Essentially, turbulent flow chromatography helps separate the proteins out from the rest of the sample, and mass spectrometry calculates the mass of each peptide, so we can specifically identify it. Scientists would know a sample was positive for SARS-CoV-2 if both viral peptides were present.
The results showed that a proteomics approach to COVID-19 diagnostics could process more than 500 samples in the span of 24 hours for approximately half the cost of the standard real-time RT-PCR test. While these tests are subject to disruption by mutation of the virus, as are the PCR tests, Cardozo et al. noted that as a positive case is indicated by the presence of both peptides, so should a mutation occur in either, the presence of only one peptide would lead to an attempt at verification by PCR. By tweaking the TFC-MS/MS process they were able to increase the speed of analysis and minimize carryover — the contamination of samples from products left over in the instrument — to a significant degree. They also found that the protein samples were easier to manage than RNA, able to sit at room temperature in saline for up to 30 days while RNA must be stored at -80℃. Furthermore, they confirmed that the results are unaffected when the sample is subjected to thermal inactivation, so it will be safer conducting these tests in a laboratory without the risk of infection. However, their final results indicated a specificity of 93.3% and a sensitivity of only 83.6%, meaning 16.4% of positive cases would incorrectly test negative, and this method is not as effective as the current PCR test. The researcher’s acknowledged that currently no other protein analysis could beat PCR’s sensitivity, and attributed this to the fact that proteins cannot be copied, so there is less material to work with compared to the multiple copies of RNA generated by the PCR test. While the prospect of using proteomics for viral diagnostics is certainly exciting, and appears to be fast, less expensive, and better at processing large batches of samples then any other current method, it doesn’t make much sense to use proteomics to test for the coronavirus when the test’s accuracy still does not match that of the “gold standard” PCR. The researchers clarify that rather than replacing PCR, this new approach might help in the event of overwhelming demand for testing that constrains our ability to test via PCR. For example, if there are not enough of the required reagents or instruments available to meet testing needs. Rather than a replacement or even an alternative testing option, this approach is better suited to a second rank choice, to be used only in dire need. However the amount of effort it would take to standardize this process so it could be widely used does not seem worth it, especially when we are currently experiencing a lull in the effects of the pandemic where there is no such emergent demand for rapid testing. And, the paper notes that like PCR, MS also relies on instruments and reagents that might be subject to limited availability, diminishing its potential usefulness. Still, these results are very promising, and the proof of concept could lead to new research for other viral diagnostic processes in the future.
This is where
Cardozo K. H. M., A. Lebkuchen, G. G. Okai, R. A. Schuch, L. G. Viana, et al., 2020 Establishing a mass spectrometry-based system for rapid detection of SARS-CoV-2 in large clinical sample cohorts. Nat. Commun. 11: 6201. https://doi.org/10.1038/s41467-020-19925-0
Corman V. M., O. Landt, M. Kaiser, R. Molenkamp, A. Meijer, et al., 2020 Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Eurosurveillance 25: 2000045. https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045
COVID-19 RT-PCR Test – EUA Summary, 2021 30.
Esbin M. N., O. N. Whitney, S. Chong, A. Maurer, X. Darzacq, et al., 2020 Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection. RNA N. Y. N 26: 771–783. https://doi.org/10.1261/rna.076232.120
Isabelle Wierum is a junior Bioinformatics major at Davidson College. Contact her at iswierum@davidson.edu.
© Copyright 2020 Department of Biology, Davidson College, Davidson, NC 28036.
