Intact-Mass Analysis Facilitating the Identification of Large Human Heart Proteoforms

Proteoforms, the primary effectors of biological processes, are the different forms of proteins that arise from molecular processing events such as alternative splicing and post-translational modifications. Heart diseases exhibit changes in proteoform levels, motivating the development of a deeper understanding of the heart proteoform landscape. Our recently developed two-dimensional top-down proteomics platform coupling serial size exclusion chromatography (sSEC) to reversed-phase chromatography (RPC) expanded coverage of the human heart proteome and allowed observation of high molecular weight proteoforms. However, most of these observed proteoforms were not identified due to the difficulty in obtaining quality tandem mass spectrometry (MS2) fragmentation data for large proteoforms from complex biological mixtures on a chromatographic time scale. Herein, we sought to identify human heart proteoforms in this data set using an enhanced version of Proteoform Suite, which identifies proteoforms by intact mass alone. Specifically, we added a new feature to Proteoform Suite to determine candidate identifications for isotopically unresolved proteoforms larger than 50 kDa, enabling subsequent MS2 identification of important high-molecular weight human heart proteoforms such as lamin A (72 kDa) and trifunctional enzyme subunit alpha (79 kDa). With this new workflow for large proteoform identification, endogenous human cardiac myosin binding protein C (140 kDa) was identified for the first time. This study demonstrates the integration of our sSEC-RPC-MS proteomics platform with intact-mass analysis through Proteoform Suite to create a catalog of human heart proteoforms and facilitate the identification of large proteoforms in complex systems.