Currently, more than 20,000 diseases are affecting human health. Out of these, only a minor proportion of diseases have specific and accurate diagnostic tests. Moreover, in the case of diseases such as diabetics, well-established diagnostic tests can be further supported with novel prognostic biomarkers.

Biomarkers capable of detecting early stages can prove beneficial in understanding disease mechanisms and paving the way for newer and more effective therapeutic interventions. Moreover, accurate biomarkers are becoming necessary for current conditions such as inflammatory bowel disease. Similarly, novel accurate biomarkers can become crucial in evaluating clinical trial outcomes in the context of disease progression or treatment outcomes.

An LC-MS-based analysis is a powerful way for discovering and analyzing new disease biomarkers. However, some crucial parameters must be controlled and assessed during experimental research and execution. Today's article discusses the critical technical aspects of LC-MS assays in biomarker discovery.

Key pointer for LC-MS analysis in biomarker discovery

A typical biomarker development process is divided into three stages; discovery, verification, and validation. The validation phase is further segregated into analytical validation and clinical validation. Clinical validation is often called the qualification phase.

Researchers focus on identifying several candidate biomarkers in the discovery phase. They primarily conduct in-depth untargeted proteomic analysis to discover and estimate as many peptides as possible in the early stages. These proteins, thereupon, lead to the identification of numerous potential biomarker candidates that can be later assessed in the verification and validation phases. However, due to logistics and cost limitations, only a limited number of biomarkers are selected for analysis.

Researchers use LC-MS/MS analysis to predict the MS/MS spectra of putative biomarkers. Although this process is quite early in the discovery phase, the biomarkers selected are putative peptide markers. And therefore, they should be verified in similar samples to be used in subsequent discovery phases.

As outlined earlier, the primary goal of the biomarker discovery phase is to identify as many biomarkers as possible early in the discovery phase. Hence, bioanalytical scientists use LC-MS/MS analysis to perform in-depth proteomics with a limited number of potential biomarker candidates. Here the main aim is to cover as much depth as possible of the proteome. Thus researchers assess sample complexity and employ peptide prefractionation and abundant protein depletion to ensure a maximum number of proteins present at low levels are detected. Furthermore, they often use peptides labeled with isobaric tags for multiplexing samples, decreasing the chances of variability among different measurements.

Conclusion

Advancements in MS systems are crucial for biomarker discovery and development. However, it will be critical how bioanalytical scientists assess and overcome challenges associated with LC-MS method validation. Today low throughput and high data collection costs are new challenges in LC-MS analysis. Nonetheless, multiplexing with isobaric tags and faster and efficient chromatography techniques have drastically curbed these shortcomings. Even with such crucial advances in HPLC analysis, effective LC-MS/MS method development and validation will be the key for its long-term use in biomarker discovery.