Native Proteomics: A New Approach to Protein Complex Discovery and Characterization
Authors: Compton Philip Daniel , Skinner Owen , Haverland Nicole , Fornelli Luca , Doubleday Peter , Seckler Henrique , Schachner Luis , Kelleher Neil 
Published: June 2021

Protein complexes play critical roles in an array of biological processes, including cancer, senescence, and cell cycle arrest. In order to perform these functions, protein complexes exhibit great diversity in protein membership, post‐translational modification and noncovalent cofactors. Given the vital functions and varied composition of these macromolecular assemblies, their identification and structural characterization is foundational to our understanding of normal and disease biology. Thus, examination of cellular products at progressively higher levels of complexity, from peptides to proteins to protein complexes, paints an increasingly complete picture of the actuators of biological systems.Current methodologies for analysis of protein complexes fall into two groups: those that provide detailed characterization of a single target and those that provide low‐level information on a large number of interactions. Examples of the former include X‐ray crystallography, fluorescence imaging, cryo‐electron microscopy, and co‐immunoprecipitation, which are lower‐throughput techniques that can require large amounts of purified and optimized samples. In contrast, the latter category includes the use of tandem mass spectrometry (MS) techniques in a peptide‐based “bottom‐up” methodology. While bottom‐up approaches can provide extensive maps of physical interactions, they often fail to determine stoichiometry or the complete modification states of complex subunits.In this contribution, we describe the first large‐scale study utilizing our new Native Proteomics platform that occupies the space between existing approaches to gain complete molecular detail from relatively small amounts of unpurified endogenous protein complexes. Native proteomics aims to directly identify and characterize proteoforms and the complexes generated from them, which we term multi proteoform complexes (MPCs). Beyond sample preparation and separation strategies that retain native protein structure, the key to Native Proteomics is a 3‐tiered tandem mass spectrometric approach. During this experiment, a single charge state of one intact MPC is isolated and activated to eject one or multiple subunits. Next, activation is performed prior to isolation and each of the ejected monomers is isolated and fragmented individually. This approach yields an intact mass for the entire protein complex, intact masses of the ejected subunits, and backbone fragment ion masses from each fragmented subunit. Thus, in a single experiment, an unknown complex may be identified by its constituent subunits, stoichiometry may be determined from the intact mass coupled with the knowledge of subunit masses, and any additional sources of mass, such as cofactors and other PTMs, can be identified.In our initial dataset, we identified and characterized 125 unique protein complexes ranging in size from 5–316 kDa. Within these complexes, 28 exhibited metal binding events, 2 of which were previously unannotated in UniProt. Further, we characterized small molecule binding events, covalent cofactors, numerous unannotated PTMs on cysteine side chains, endogenous cleavages and 2 examples of stoichiometric regulation of complex assembly. The level of molecular detail that this platform achieves on endogenous material is unparalleled and represents a new strategy that, for the first time, enables protein complexes to be the primary analytical targets in proteomics research.Support or Funding InformationFunding for this project was provided by the W. M. Keck foundation (DT061512) and Northwestern University. OSS and PFD are supported by US National Science Foundation graduate research fellowships (2014171659 and 2015210477, respectively). 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.