Mass Spectrometry Approaches in Protein Structural Analysis

Mass spectrometry has evolved into a cornerstone of structural proteomics, offering rapid, sensitive and site-specific insights into protein architecture, interactions and dynamics. By coupling chemical labelling or cross-linking strategies with high-resolution mass analysis, researchers can probe solvent accessibility, conformational changes and binding interfaces without requiring crystallisation or extensive sample preparation. Techniques such as hydrogen–deuterium exchange report on backbone flexibility, while hydroxyl radical footprinting and covalent labelling reveal side-chain exposure and ligand-induced alterations. Native mass spectrometry preserves non-covalent assemblies, enabling mass measurement of intact complexes and subunit stoichiometry. Integrative frameworks that merge sparse mass spectrometry restraints with computational modelling and artificial-intelligence predictions now permit the generation and evaluation of atomic-resolution models. These advances underpin applications in drug development, antibody engineering, membrane-protein characterisation and fundamental studies of folding pathways, thus highlighting the global impact of mass spectrometry in deciphering protein structure and function.

Research from Nature Portfolio

Recent studies have demonstrated the power of combining covalent-labelling mass spectrometry with machine-learning–based modelling to resolve complex assembly structures at near-atomic resolution. In one approach, differential labelling yields from photo-activated probes were integrated into docking simulations of multi-domain proteins, distinguishing native interfaces with root-mean-square deviations below 4 Å. Another work introduced a dynamics-driven hydroxyl radical footprinting pipeline that directs ab initio structure prediction: time-resolved solvent exposure data were incorporated into scoring functions, markedly improving the accuracy of low-energy models and facilitating reliable selection of atomic-detail structures for benchmark proteins.

Mass Spectrometry Approaches in Protein Structural Analysis publication trend

The graph below shows the total number of articles in mass spectrometry approaches in protein structural analysis across all publications each year (not limited to Nature Index journals).

Technical terms

Covalent labelling mass spectrometry: A technique in which reactive probes form covalent bonds with solvent-exposed amino acid side chains, enabling their identification by mass analysis to infer protein surface topology.

Fast photochemical oxidation of proteins (FPOP): A footprinting approach generating hydroxyl radicals via laser-induced hydrogen peroxide photolysis, irreversibly tagging solvent-accessible residues on a microsecond timescale.

Hydroxyl radical footprinting: An irreversible labelling method using hydroxyl radicals to oxidise side chains in solution, reporting on local solvent accessibility and conformational changes.

Hydrogen–deuterium exchange (HDX): A reversible labelling technique measuring the exchange of backbone amide hydrogens with deuterium in solution, reflecting hydrogen-bond stability and protein dynamics.

Carbene footprinting: A photo-activated covalent-labelling strategy employing carbene species to map protein–ligand or protein–protein interaction sites with rapid and efficient tagging.

Solvent accessible surface area (SASA): The surface area of a protein or residue accessible to solvent molecules, often inferred from labelling extents or computational models to gauge exposure.

References

1. Site-Specific Structural Changes in Long-Term-Stressed Monoclonal Antibody Revealed with DEPC Covalent-Labeling and Quantitative Mass Spectrometry. Pharmaceuticals (2023).
2. Quantifying the Impact of the Peptide Identification Framework on the Results of Fast Photochemical Oxidation of Protein Analysis. Journal of Proteome Research (2023).
3. Protein Footprinting Comes of Age: Mass Spectrometry for Biophysical Structure Assessment*. Molecular & Cellular Proteomics (2017).
4. Fast photochemical oxidation of proteins (FPOP): A powerful mass spectrometry–based structural proteomics tool. Journal of Biological Chemistry (2019).
5. Carbene footprinting accurately maps binding sites in protein–ligand and protein–protein interactions. Nature Communications (2016).
6. Quantitative Protein Topography Measurements by High Resolution Hydroxyl Radical Protein Footprinting Enable Accurate Molecular Model Selection. Scientific Reports (2017).
7. Surface Accessibility and Dynamics of Macromolecular Assemblies Probed by Covalent Labeling Mass Spectrometry and Integrative Modeling. Analytical Chemistry (2017).
8. Accurate protein structure prediction with hydroxyl radical protein footprinting data. Nature Communications (2021).
9. Protein complex prediction using Rosetta, AlphaFold, and mass spectrometry covalent labeling. Nature Communications (2022).