Abstract
The term "proteoforms" describes the range of different structures of a protein product of a single gene, including variations in amino acid sequence and post-translational modifications. This diversity in protein structure contributes to the biological complexity observed in living organisms. As the concentration of a particular proteoform may increase or decrease in abnormal physiological states, proteoforms have long been used in medicine as biomarkers of health and disease. Notably, the analytical approaches used to analyze proteoforms have evolved considerably over the years. While ligand binding methods continue to play a large role in proteoform measurement in the clinical laboratory, unanticipated or unknown post-translational modifications and sequence variants can upend even extensively tested and vetted assays that have successfully made it through the medical regulatory process. As an alternate approach, mass spectrometry-with its high molecular selectivity-has become an essential tool in detection, characterization, and quantification of proteoforms in biological fluids and tissues. This review explores the analytical techniques used for proteoform detection and quantification, with an emphasis on mass spectrometry and its various applications in clinical research and patient care including, revealing new biomarker targets, helping improve the design of contemporary ligand binding in vitro diagnostics, and as mass spectrometric laboratory developed tests used in routine patient care.