Dissecting the structural heterogeneity of proteins by native mass spectrometry

Abstract

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.

Protein structure and dynamics in the era of integrative structural biology

“…Native MS provides species distributions of conformers and supramolecular complexes upon gentle desolvation and ionization, yielding values of stoichiometry and solvent accessible surface area (Tamara et al, 2022). Combination with IM introduces an additional dimension in ion separation and analysis, detecting structural heterogeneity even within each charge state and measuring rotationally-averaged, collisional cross-section (CCS) (Kaltashov et al, 2022;Santambrogio et al, 2022, Christofi andBarran, 2023;Reid et al, 2023).…”

“…This rich structural information can guide particle classification in cryo-EM/ET, ensemble deconvolution by NMR and computational modelling by experimental constraints. Structural heterogeneity deriving from PTMs, ligand binding and protein assemblies can also be described (Kaltashov et al, 2022;Reid et al, 2023). MS methods can in principle be implemented in a time-resolved mode on the millisecond scale (even microsecond for FPOP), although such instrumental setups are still not widespread (Lento and Wilson, 2022).…”

Mass Spectrometry-Based Proteomic Technology and Its Application to Study Skeletal Muscle Cell Biology

“…The rapid developments of novel proteomic technologies and applications has led to the establishment of many specialized approaches focusing on targeted biomarker proteomics [63,64], clinical proteomics [65,66], drug discovery and pharmaco-proteomics [67][68][69], proteogenomics [70,71], membrane proteomics [72,73], extracellular matrix proteomics [74,75], native proteomics [76][77][78], thermal proteome profiling [79], the analysis of protein conformations and interactions via chemical cross-linking MS [80,81] and subcellular/organelle proteomics [82,83]. The more recently developed discipline of native MS focuses on the detailed top-down characterization of structural heterogeneity in protein isoforms under non-denaturing conditions, making this approach an important part of structural cell biology [84,85].…”

Hydrophilic interaction chromatography coupled to ultraviolet photodissociation affords identification, localization, and relative quantitation of glycans on intact glycoproteins

“…24,25 However, few studies have reported top-down analysis of glycoproteins, which requires advanced activation techniques, 26 advanced separation techniques 27 , or may utilize partial enzymatic deglycosylation [28][29][30] to mitigate the analytical challenge presented by glycan heterogeneity which creates an overlap of glycoproteoforms in the m/z domain. 31 One of the major ongoing challenges in the top-down proteomics workflow is the successful separation of glycoproteins. Traditional reversed phase liquid chromatography (RPLC) methods (C18, C4, PLRP, and others) are suitable for the separation of cleaved permethylated glycans, 32 small glycopeptides, [33][34][35] and some intact proteins, 21 but do not always provide sufficient resolution of larger glycans and glycosylated proteins.…”