Integrated Top-Down Proteomics for Comprehensive Proteome Analysis at the Proteoform Level

Background: Proteoforms are the critical functional species that drive biological processes. However, conventional proteogenomic (“shotgun”) analyses discard relevant proteoform-level information through bulk protein digestion. This study presents a proof-of-principle integrative Top-Down Proteomics workflow designed to identify and characterise proteoforms across the breadth of a proteome. Methods: This workflow combined two-dimensional electrophoresis, a custom grid-cutting lattice for reproducible gel sectioning, in-gel digestion, liquid chromatography–tandem mass spectrometry, and open-search algorithms. Gel-grid locations, isoelectric points, and molecular weights were integrated with mass-spectrometry results to further characterise identified proteoforms. Results: The approach enabled identification of a broad range of proteoforms and their inherent post-translational modifications, including phosphorylation, acetylation, methylation, and glycosylation. Notably, substantial numbers of proteoforms were identified in visually “blank regions” of replicate two-dimensional gels. This eliminated bias associated with assessing only visually detectable protein spots and substantially expanded proteome-level proteoform detection and characterisation. Conclusions: This integrative workflow provides a reproducible and comprehensive strategy for addressing the often-overlooked complexity of proteomes at the proteoform level. By extending analysis beyond visually detectable gel spots, it enables deeper characterisation of proteoform diversity and associated post-translational modifications.