Hydrogen–Deuterium Exchange Mass Spectrometry with Integrated Size-Exclusion Chromatography for Analysis of Complex Protein Samples

The growing use of hydrogen–deuterium exchange mass spectrometry (HDX-MS) for studying membrane proteins, large protein assemblies, and highly disulfide-bonded species is often challenged by the presence in the sample of large amounts of lipids, protein ligands, and/or highly ionizable reducing agents. Here, we describe how a short size-exclusion chromatography (SEC) column can be integrated with a conventional temperature-controlled HDX-MS setup to achieve fast and online removal of unwanted species from the HDX sample prior to chromatographic separation and MS analysis. Dual-mode valves permit labeled proteins eluting after SEC to be directed to the proteolytic and chromatographic columns, while unwanted sample components are led to waste. The SEC-coupled HDX-MS method allows analyses to be completed with lower or similar back-exchange compared to conventional experiments. We demonstrate the suitability of the method for the analysis of challenging protein samples, achieving efficient online removal of lipid components from protein–lipid systems, depletion of an antibody from an antigen during epitope mapping, and elimination of MS interfering compounds such as tris­(2-carboxyethyl)­phosphine (TCEP) during HDX-MS analysis of a disulfide-bonded protein. The implementation of the short SEC column to the conventional HDX-MS setup is straightforward and could be of significant general utility during the HDX-MS analysis of complex protein states.