Mass Spectrometry for Biomarker Discovery: Pros and Cons

The biomedical field is currently plagued with a reproducibility crisis. Alarmingly, it has been estimated that over 50% of all research findings cannot be replicated. Because antibodies often display nonspecific binding and may experience lot-to-lot variability, it has been suggested that antibody-based methods may be contributing to this reproducibility crisis.

One advantage of using mass spectrometry for biomarker identification is its high accuracy and specificity. However, mass spectrometry tends to be slower than many high-throughput approaches involving antibodies. Biocompare recently interviewed experts from four companies (RayBiotech, Sapient, Thermo Fisher Scientific, and Waters Corporation) to explore the advantages and disadvantages of mass spectrometry for biomarker research.

From discovery to validation

Biomarkers must first be discovered and then validated before they can be used in clinical trials. The discovery process―typically with either mass spectrometry or large-scale multiplex antibody assays—involves the initial screening of a small number of samples for thousands of markers. As Jarad Wilson, Associate Director of Business Development at RayBiotech, describes the discovery process, “You throw enough wet spaghetti and hope something will stick.”

In contrast to discovery, validation involves analyzing a smaller subset of identified markers—typically one to ten—in a much larger number of samples. Dr. Mo Jain, Founder and CEO of Sapient, explains that validation can be broadly broken down into two stages: technical validation and biological validation.

Technical validation involves using alternative bioanalytical technologies—often referred to as orthogonal methods—to confirm measurements. For example, if you use mass spectrometry for discovery, validation may follow up with multiplex arrays, western blots, immunohistochemistry, or single-target ELISA.

On a broader level, biological validation is necessary to assess how your biomarker of interest behaves across different individuals and diverse populations. The process of biological validation often requires hundreds to thousands of specimens. Ultimately, the goal is to ensure that only the most biologically relevant biomarkers are being identified—thus minimizing false discoveries.

Why mass spectrometry for biomarker discovery?

Santosh Renuse, Ph.D., Senior Marketing Manager for Thermo Fisher Scientific, emphasizes that one of the main advantages of mass spectrometry for protein biomarker discovery is its high accuracy and specificity. On the downside, Renuse notes that mass spectrometry is prone to dynamic range problems in serum and plasma samples. After all, concentrations of different proteins often vary by more than ten orders of magnitude.

Mass spectrometry also tends to be somewhat slower than many multiplex antibody-based approaches. Nevertheless, throughput has been increasing over time. Renuse notes that, in single-shot experiments, mass spectrometry is now sensitive enough to detect over 7,000 proteins from cells and tissues in an hour. This constitutes over half of the expressed proteome.

Finally, mass spectrometers can be expensive and difficult to operate correctly. Wilson points out that RayBiotech, which has successfully used its multiplex sandwich antibody arrays to develop quantitative assays for over 1600 human biomarkers, is evaluating whether to incorporate mass spectrometry. “Newer mass spec systems have gotten smaller in past years, and the price has come down some, but there is certainly a high upfront cost to get this set up,” he notes.

Discovery with mass spectrometry

Renuse highlights several techniques that involve mass spectrometry for the discovery process. For instance, common label-based quantitative methods include stable isotope labeling with amino acids in cell culture (SILAC) and tandem mass tags (TMT).

Label-free methods are also often used for quantification in biomarker discovery studies. These approaches include label-free quantitation (LFQ) and data-independent acquisition (DIA). In particular, LFQ is becoming popular for biomarker discovery studies because it is inexpensive and allows for the comparative analysis of large samples. However, LFQ also tends to be less accurate than many label-based methods.

State-of-the-art advances in mass spec for biomarker research

Jain emphasizes that Sapient is focusing on the discovery of small molecule biomarkers―as opposed to larger proteins—using what they call rapid liquid chromatography–mass spectrometry (rLC-MS). He explains, “We developed these technologies to run high-throughput, nontargeted discovery screenings on diverse biological specimens,” noting that circulating small molecule chemistry reveals the non-genetic landscape of disease.

Using rLC-MS for discovery, Sapient can measure over 11,000 small molecule biomarkers per biological sample. Sapient’s analysis captures known and unknown molecules across broad chemistries, ranging from very polar to very nonpolar molecules.

“Because our approach is nontargeted, the vast majority of the biomarkers we capture are unknown, meaning they have never been measured or structurally elucidated,” Jain points out. The rLC-MS method uses ion mobility spectrometry (IMS) to chemically fingerprint thousands of unknown markers. More specifically, Sapient uses retention time, accurate mass, and collisional cross-section (CSS) to identify and classify each unknown molecule.

Joseph Fredette, Principal Product Manager of Consumables and Lab Automation at Waters Corporation, agrees with Jain that IMS is playing a pivotal role in biomarker discovery. Fredette highlights Waters’ SELECT SERIES Cyclic IMS time-of-flight mass spectrometer, which uses novel cyclic ion mobility to separate biological molecules based on their size and weight (mass-to-charge ratio) as well as their shape. As he explains, “Isomeric protein variants that fold differently and have different shapes can be separated using IMS instruments like these, and this added separation power can be very powerful.”

The future

Clinical trials with biomarkers are two to ten times more likely to obtain drug approval. Furthermore, with its high sensitivity, mass spectrometry may have the potential to overcome some of the reproducibility concerns associated with antibody-based approaches. And while mass spectrometry tends to be slower than many antibody-based methods, throughput is rapidly improving. Jain concludes: “Now that we’ve begun to release the brakes for greater speed, I think we are just at the start of leveraging everything that mass spectrometry can actually do.”