Native Mass Spectrometry at the Convergence of Structural Biology and Compositional Proteomics.

Biology is driven by a vast set of molecular interactions that evolved over billions of years. Just as covalent modifications like acetylations and phosphorylations can change a protein's function, so too can noncovalent interactions with metals, small molecules, and other proteins. However, much of the language of protein-level biology is left either undiscovered or inferred, as traditional methods used in the field of proteomics use conditions that dissociate noncovalent interactions and denature proteins.Just in the past few years, mass spectrometry (MS) has evolved the capacity to preserve and subsequently characterize the complete composition of endogenous protein complexes. Using this "native" type of mass spectrometry, a complex can be activated to liberate some or all of its subunits, typically via collisions with neutral gas or solid surfaces and isolated before further characterization ("Native Top-Down MS," or nTDMS). The subunit mass, the parent ion mass, and the fragment ions of the activated subunits can be used to piece together the precise molecular composition of the parent complex. When performed en masse in discovery mode (i.e., "native proteomics"), the interactions of life─including protein modifications─will eventually be clarified and linked to dysfunction in human disease states.In this Account, we describe the current and future components of the native MS toolkit, covering the challenges the field faces to characterize ever larger bioassemblies. Each of the three pillars of native proteomics are addressed: (i) separations, (ii) top-down mass spectrometry, and (iii) integration with structural biology. Complexes such as endogenous nucleosomes can be targeted now using nTDMS, whereas virus particles, exosomes, and high-density lipoprotein particles will be tackled in the coming few years. The future work to adequately address the size and complexity of mega- to gigadalton complexes will include native separations, single ion mass spectrometry, and new data types. The use of nTDMS in discovery mode will incorporate native-compatible separation techniques to maximize the number of proteoforms in complexes identified. With a new wave of innovations, both targeted and discovery mode nTDMS will expand to include extremely scarce and exceedingly heterogeneous bioassemblies. Understanding the proteinaceous interactions of life and how they go wrong (e.g., misfolding, forming complexes in dysfunctional stoichiometries and configurations) will not only inform the development of life-restoring therapeutics but also deepen our understanding of life at the molecular level.