Top-Down Mass Spectrometry-Based Structural and Functional Proteomics: A Perspective

A wide range of protein modifications (e.g., truncations, amino acid substitutions, and posttranslational modifications, PTMs) create diverse proteoforms that govern physiological regulation and support cellular homeostasis. As such, understanding proteoform structure is fundamental to elucidating biological function and disease mechanisms. Traditional high-resolution methods (e.g., NMR, cryo-EM, and X-ray crystallography) offer powerful structural information but remain limited in their capacity to interrogate the structural effects of proteoform heterogeneity. Mass spectrometry (MS)-based structural proteomics allows the evaluation of protein structure in native and native-like conditions. Typically, MS-based structural proteomics methods implement bottom-up proteomics in which proteins are digested into small peptides prior to MS detection. This digestion, however, may obscure proteoform structural information such as coordinating PTMs. Top-down proteomics, on the other hand, analyzes intact proteoforms directly to preserve the connectivity between proteoform structure and function. In this perspective, we discuss the application of top-down MS-based proteomics for interrogating intact proteoform structures and outline future directions for the field. We highlight the potential of top-down structural proteomics as a powerful and complementary approach to bottom-up proteomics and traditional biochemical strategies, enabling comprehensive characterization of the intact structural proteome.