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 using 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 MS 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. These data enable precise compositional analysis of protein complexes as they exist in the cell and demonstrate a new approach that uses MS as a bridge to structural biology.
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Funding for this project was provided by the W.M. Keck foundation (DT061512) and Northwestern University. This research was also supported by the Paul G. Allen Family Foundation (Grant Award 11715). The authors would also like to acknowledge helpful discussion with A. Catherman, R. Fellers, J. Greer, and B. Early. Helpful assistance from Thermo Fisher Scientific was provided by M. Belov, S. Horning, and A. Makarov. OSS and PFD are supported by US National Science Foundation graduate research fellowships (2014171659 and 2015210477, respectively). LHFDV is supported under CNPq research grant 400301/2014-8 from the Brazilian government. LFS was supported by the Chemistry of Life Processes Predoctoral training program at Northwestern University. Additional support for the maintenance of the SEMPC from the National Resource for Translational and Developmental Proteomics (GM108569) is acknowledged.
N.L.K. serves as a paid consultant to Thermo Fisher Scientific, whose instruments were used in this work.
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Skinner, O., Haverland, N., Fornelli, L. et al. Top-down characterization of endogenous protein complexes with native proteomics. Nat Chem Biol 14, 36–41 (2018). https://doi.org/10.1038/nchembio.2515
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