Protein complexes exhibit great diversity in protein membership, post-translational modifications and noncovalent cofactors, enabling them to function as the actuators of many important biological processes. The exposition of these molecular features with current methods lacks either throughput or molecular specificity, ultimately limiting the use of protein complexes as direct analytical targets in a wide range of applications. Here, we apply native proteomics, enabled by a multistage tandem mass spectrometry approach, to characterize 125 intact endogenous complexes and 217 distinct proteoforms derived from mouse heart and human cancer cell lines in discovery mode. The native conditions preserved soluble protein–protein interactions, high-stoichiometry noncovalent cofactors, covalent modifications to cysteines, and, remarkably, superoxide ligands bound to the metal cofactor of superoxide dismutase 2. The data enable precise compositional analysis of protein complexes as they exist in the cell and demonstrate a new approach that uses mass spectrometry as a bridge to structural biology.

Efforts to map the human protein interactome have resulted in information about hundreds to thousands of multi-protein assemblies housed in public repositories, but the molecular characterization and stoichiometry of their protein subunits remains largely unknown. Here, we combined the CORUM and UniProt databases to create candidates for an error-tolerant search engine designed for hierarchical top-down analyses, identification, and scoring of multi-proteoform complexes by native mass spectrometry.

The cadre of protein complexes in cells performs an array of functions necessary for life. Their varied structures are foundational to their ability to perform biological functions, lending great import to the elucidation of complex composition and dynamics. Native separation techniques that are operative on low sample amounts and provide high resolution are necessary to gain valuable data on endogenous complexes. Here, we detail and optimize the use of tube gel separations to produce samples proven compatible with native, multistage mass spectrometry (nMS/MS). We find that a continuous system (i.e., no stacking gel) with a gradient in its extent of cross-linking and use of the clear native buffer system performs well for both fractionation and native mass spectrometry of heart extracts and a fungal secretome. This integrated advance in separations and nMS/MS offers the prospect of untargeted proteomics at the next hierarchical level of protein organization in biology.