Dissecting the structural heterogeneity of proteins by native mass spectrometry

A single gene yields many forms of proteins via combinations of posttranscriptional/posttranslational modifications. Proteins also fold into higher‐order structures and interact with other molecules. The combined molecular diversity leads to the heterogeneity of proteins that manifests as distinct phenotypes. Structural biology has generated vast amounts of data, effectively enabling accurate structural prediction by computational methods. However, structures are often obtained heterologously under homogeneous states in vitro. The lack of native heterogeneity under cellular context creates challenges in precisely connecting the structural data to phenotypes. Mass spectrometry (MS) based proteomics methods can profile proteome composition of complex biological samples. Most MS methods follow the “bottom‐up” approach, which denatures and digests proteins into short peptide fragments for ease of detection. Coupled with chemical biology approaches, higher‐order structures can be probed via incorporation of covalent labels on native proteins that are maintained at the peptide level. Alternatively, native MS follows the “top‐down” approach and directly analyzes intact proteins under nondenaturing conditions. Various tandem MS activation methods can dissect the intact proteins for in‐depth structural elucidation. Herein, we review recent native MS applications for characterizing heterogeneous samples, including proteins binding to mixtures of ligands, homo/hetero‐complexes with varying stoichiometry, intrinsically disordered proteins with dynamic conformations, glycoprotein complexes with mixed modification states, and active membrane protein complexes in near‐native membrane environments. We summarize the benefits, challenges, and ongoing developments in native MS, with the hope to demonstrate an emerging technology that complements other tools by filling the knowledge gaps in understanding the molecular heterogeneity of proteins.