Mapping the Proteoform Landscape of Five Human Tissues

A functional understanding of the human body requires structure- function studies of proteins at scale. The chemical structure of proteins is controlled at the transcriptional, translational, and post-translational levels, creating a variety of products with modulated functions within the cell. The term proteoform encapsulates this complexity at the level of chemical composition. Comprehensive mapping of the proteoform landscape in human tissues necessitates analytical techniques with increased sensitivity and depth of coverage. Here, we took a top-down proteomics approach, combining data generated using capillary zone electrophoresis (CZE) and nanoflow reversed-phase liquid chromatography (RPLC) hyphenated to mass spectrometry to identify and characterize proteoforms from the human lungs, heart, spleen, small intestine, and kidneys. CZE and RPLC provided complementary post-translational modification and proteoform selectivity, thereby enhancing the overall proteome coverage when used in combination. Of the 11,466 proteoforms identified in this study, 7373 (64%) were not reported previously. Large differences in the protein and proteoform level were readily quantified, with initial inferences about proteoform biology operative in the analyzed organs. Differential proteoform regulation of defensins, glutathione transferases, and sarcomeric proteins across tissues generate hypotheses about how they function and are regulated in human health and disease.

Automated summary

To understand the human body's functions, it is necessary to study the relationship between protein structure and function on a large scale. Proteins' chemical structure is regulated at multiple levels, resulting in various products with different functions within cells. The term "proteoform" encompasses the complexity of chemical composition. Mapping the proteoform landscape in human tissues requires sensitive and comprehensive analytical techniques. In this study, a top-down proteomics approach combining capillary zone electrophoresis and nanoflow reversed-phase liquid chromatography was used to identify and characterize proteoforms from different organs. The combination of these techniques provided enhanced coverage of post-translational modifications and proteoforms. Among the identified proteoforms, a significant percentage had not been reported before. The differential regulation of proteoforms in different organs provides insights into their function and regulation in human health and disease.