Novel techniques for the quantification of protein biomarker isoforms by liquid chromatography and mass spectrometry

Biomarkers serve crucial roles across diverse fields, aiding in clinical diagnostics by distinguishing between health and disease and by monitoring treatment effectiveness. Many biomarkers are proteins and, traditionally, ligand binding assays (LBAs) are used to quantify protein biomarkers but they face challenges due to inconsistent quality of antibody reagents, hindering accuracy and comparability between laboratories. Additionally, LBAs often fail to differentiate between protein isoforms, which limits their clinical relevance.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a robust alternative for protein biomarker analysis, offering enhanced accuracy and the ability to discern between different proteins and isoforms. Despite its potential, LC-MS/MS has its own challenges such as lower sensitivity and higher operational costs. This thesis explores the application of LC-MS/MS for protein biomarker analysis, focusing on quantifying relevant biomarker isoforms.

The various chapters describe the development and use of methods for quantifying specific biomarker isoforms, notably human growth hormone (GH) and sex hormone binding globulin (SHBG) variants. These methods show good accuracy, precision, selectivity and stability across clinically relevant concentration ranges of the isoforms. In this way, LC-MS/MS helps to clarify the role of protein biomarker isoforms which is more difficult or impossible by using LBA.

Overall, LC-MS/MS presents advantages over LBAs, particularly in discerning protein isoforms, albeit with higher costs. While LBAs remain prevalent for routine analysis, LC-MS/MS has added value for specialized investigations, to clarify ambiguous results and to offer insights into biomarker isoforms. Thus, LC-MS/MS is expected to continue its role in biomarker quantification.