How LC-MS Can Support SARS-CoV-2 Research

Despite the ongoing increase in the number of vaccinated individuals, testing still has an important role to play in the global response to the COVID-19 pandemic. PCR-based testing has so far been the gold standard, but complementary methods could help to address some of the limitations associated with the technique and further aid researchers studying SARS-CoV-2.

As part of efforts to support studies in this area, Waters recently introduced a new liquid chromatography-mass spectrometry (LC-MS) test method.* Technology Networks spoke to Kevin Wyndham, senior director of research and development at Waters , to learn more about the method, its development and how it compares to PCR.

In this interview, Kevin also explains the continued need for testing and highlights how the new method could be used to support research both during and beyond the COVID-19 pandemic.

* Waters’ SARS CoV-2 LC-MS kit is for Research Use Only (RUO) – not intended for diagnostic use.

This article includes research findings that are yet to be peer-reviewed. Results are therefore regarded as preliminary and should be interpreted as such. Find out about the role of the peer review process in research here. For further information, please contact the cited source.

Anna MacDonald (AM): Can you explain the LC-MS method and how it came about? Do you have partners in this effort?

Kevin Wyndham (KW): Early in the pandemic, Waters made an important decision to focus our attention on collaborating and supporting researchers that were working on the front lines of COVID toward advances in vaccines, therapies, and testing. We called this our COVID Innovation Response Team (IRT), which has successfully created global networks of scientists working collectively.

The earliest collaborations from this program were with two teams separated by over 7000 miles and nine time zones. Dr. Maarten Dhaenens’ team at Ghent University started early with a proteomics testing approach using LC-MS to explore peptide-based markers of the SARS-CoV-2 virus. At the same time, we had started supporting work by Dr. Leigh Anderson’s team at SISCAPA Assay Technologies around peptide enrichment workflows for COVID. 

Getting these two groups working together was seamless, as there was a high commonality in the workflows. The Ghent team used a digestive enzyme to break down the virus protein contained in positive nasal swab samples into a series of peptides. They then used LC-MS to separate the peptides and quantify specific peptides characteristic of the SARS-CoV-2 virus. The SISCAPA workflow had a similar starting process, but at the end of the workflow they introduced an enrichment step for specific peptides using monoclonal antibodies attached to magnetic beads. The use of selective antibody for the peptide of interest allowed for higher test sensitivity and very high specificities.

Over the summer months, the collaborations expanded greatly when the UK Government and National Health Services created a formal program exploring the utility of mass spectrometry for COVID testing.  This accelerated program, aptly named Operation Moonshot, brought together several leading scientific teams throughout the UK. As a result, we soon found ourselves engaging with groups from the University of Manchester, University of Leicester, University College London, University of Southampton, Imperial College London, Kings College London, Leeds University, Viapath-St Thomas’ Hospital, and the Karolinska Hospital in Sweden.

AM: How does this method differ from PCR-based approaches, which have been the traditional methods of diagnostic testing to date? 

KW: PCR-based COVID testing has been the gold standard and is both sensitive and specific. An LC-MS-based approach, which is still yet to be approved for clinical use, is best considered a complementary technique to PCR. The LC-MS technique itself has several unique differences in the way it could be used to assess the presence of SARS-CoV-2.

PCR essentially goes about amplifying viral-RNA. In this process, we can take trace concentrations of RNA and continually increase concentrations by repeating the number of amplification cycles. This allows it to be highly sensitive and have high testing thresholds. However, as noted by Dr. Mina from the Harvard School of Public Health, “tests with thresholds so high may detect not just live virus but also genetic fragments, leftovers from infection that pose no particular risk — akin to finding a hair in a room long after a person has left.”

Unlike an RNA-based test, the LC-MS testing analyzes peptides that stem from viral proteins without replication. As such, the LC-MS method may realize a direct and quantitative analysis of viral load. This is an important research tool as the viral load can potentially be correlated with the severity of illness. As this LC-MS technique is a RUO reagent, more research and development steps are needed to confirm the technique potentials.

AM: With vaccines being aggressively rolled out worldwide, what role do you see testing techniques like this one playing?

KW: The speed of development and the global impact that vaccines are making on our ability to address this pandemic is truly amazing. While we are making huge strides in combatting COVID, there will be many enduring research areas for years to come. Today, we see a new set of questions emerge around effectiveness of vaccines with new variants, and whether we will need regular booster shots.   

To address these questions, we need a series of analytical tools – PCR-tests, antigen-tests, NGS, antibody and serology testing are all essential tools. The addition of LC-MS to this toolbox both compliments these methods and adds some highly sensitive and specific new tools. LC-MS may directly assess the viral load, rather than inferring viral load through secondary methods, and will play an important role as teams explore longitudinal patient studies. As we look to the near future, LC-MS allows for multi-analyte testing, enabling researchers to explore multiple questions at once. As the science advances, we expect LC-MS will be able to identify the presence of multiple infectious diseases and health monitory of patient biomarkers, all in the same test.   

AM: What are the benefits to using this method over PCR?

KW: The use of LC-MS to study SARS-CoV-2 both compliments PCR testing and has some unique advantages. PCR testing explores the presence of viral-RNA, while our LC-MS method interrogates the presence of viral proteins. Both approaches are sensitive techniques which may be used together to study the impact of infection on patient health longitudinally, assess the severity of illness, and determine the post-infectious state.

There are differences in the results obtained from LC-MS and PCR. One of the most significant areas is potential false positives. A false positive COVID test result concludes that the person tested is sick, or infectious, when in reality, they are not. The false positive rate for PCR has been noted to be as high as 4% in routine analysis settings.

There are many reasons why PCR tests can have elevated false positives, such as laboratory testing errors, cross-contamination of samples, and reagent purity. However, as noted earlier, the PCR experiments amplify both active and inactive (fragments) of viral-RNA. This means it can result in a positive result even for patients that are post-infectious. Antigen tests for SARS-CoV-2 are generally less sensitive than real-time reverse transcription polymerase chain reaction (RT-PCR) and other nucleic acid amplification tests (NAATs) for detecting the presence of viral nucleic acid. However, NAATs can remain positive for weeks to months after initial infection and can detect levels of viral nucleic acid even when virus cannot be cultured, suggesting that the presence of viral nucleic acid may not always indicate contagiousness.      

The concern of false positives results with PCR increases when the infectious rate is decreasing. As the false positive rate is fairly constant, this error becomes more pronounced at lower positivity rates, making it hard for a test to discriminate between true and false positives. This can have significant economic and societal impacts, including loss of productivity due to unnecessary quarantines and healthcare costs of tending to healthy patients.

The Waters SARS-CoV-2 LC-MS workflow inherently has increased specificity and reduced false positives.  A recent study (clinical study reference: DNR - 2020-06395) by the Karolinska Hospital in Sweden of 88 patient samples showed 83.3% sensitivity (e.g., 8 false negatives) and 100% specificity (0 false positives).  The LC-MS-based approach has several factors that allow us to achieve highly sensitive and specific results, including high specificity antibody capture sections for three different peptide markers, stable isotope standards, automation, as well as separation in both LC and MS dimension.

When comparing PCR and LC-MS testing approaches, there are financial considerations as well. Both high sensitivity PCR and LC-MS systems are investments. While PCR is more commonly employed in routine testing in clinical laboratories, LC-MS is a more versatile analytical tool across clinical and biomedical research laboratories. The investment in an LC-MS instrument for research of COVID can be adapted to other research areas such as biomarker research and other infectious diseases. Another advantage of mass spectrometry is that you can explore multiple analytes simultaneously in a single test.

The expenses per test are also different. PCR test kits require high purity, expensive reagents. In contrast, the reagents used in this LC-MS workflow are high purity but generally less costly, allowing for a reduction in costs per analysis. The high sensitivity of LC-MS workflows may allow for exploring additional time and cost-saving approaches such as sample pooling.

AM: Can you elaborate on the company’s recently released SARS-CoV-2 LC-MS kit? Who can use it and what type of research is it intended to support?

KW: Waters’ SARS-CoV-2 LC-MS Kit is a research use only workflow designed for clinical and biomedical research laboratories that are exploring the impact of COVID-19 on patient healthcare, infectious disease research, as well as exploring the use of mass spectrometry for surveillance testing.

As many of the research laboratories have expertise in LC-MS and are familiar with this type of protein digestion workflow, there is a lot of interest in exploring it. For groups that are less familiar with this workflow, we have an automated solution using our Andrew+ lab robotics system.

AM: Do you see the LC-MS method being used beyond the COVID-19 pandemic?

KW: Yes, there are many opportunities. The approach we explored for SARS-CoV-2 may be adaptable to the analysis of different viral proteins and biomarker studies. We are just starting this journey of using LC-MS in the clinical sciences of infectious diseases. We are learning about new ways our customers want to explore and research this technology. 

This high level of interest isn’t surprising as LC-MS is practically ubiquitous – researchers see the value in further exploring this technology in clinical sciences. Many groups were already using MS for biomarker research. The addition of a SARS-CoV-2 test allows for new tools in exploring impacts on patient health.  Another advantage of LC-MS is multi-analyte testing. While our current test analyzes SARS-CoV-2, we see interest in expanding this to create more comprehensive panels of tests.  

One crucial area of research is Long-COVID. To explore this, we want to expand the workflow to examine other relevant biomarkers. A recent study examined healthcare records for almost 2 million patients in the US that had COVID. Over 20% experienced post-COVID health concerns – including “pain, breathing difficulties, hyperlipidemia, malaise and fatigue, and hypertension.”

Today we focus on COVID-19 – the most profound global pandemic in our lifetimes. As we go forward, we aim to further explore the power of LC-MS in infectious diseases, clinical sciences, and pandemic preparedness and surveillance.

Kevin Wyndham was speaking to Anna MacDonald, Science Writer for Technology Networks.