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Abstract
NMR studies and X-ray crystallography have shown that the structures of the 99-residue amyloidogenic protein β2-microglobulin (β2m) and its more aggregation-prone variant, D76N, are indistinguishable, and hence, the reason for the striking difference in their aggregation propensities remains elusive. Here, we have employed two protein footprinting methods, hydrogen–deuterium exchange (HDX) and fast photochemical oxidation of proteins (FPOP), in conjunction with ion mobility-mass spectrometry, to probe the differences in conformational dynamics of the two proteins. Using HDX-MS, a clear difference in HDX protection is observed between these two proteins in the E-F loop (residues 70–77) which contains the D76N substitution, with a significantly higher deuterium uptake being observed in the variant protein. Conversely, following FPOP-MS only minimal differences in the level of oxidation between the two proteins are observed in the E–F loop region, suggesting only modest side-chain movements in that area. Together the HDX-MS and FPOP-MS data suggest that a tangible perturbation to the hydrogen-bonding network in the E–F loop has taken place in the D76N variant and furthermore illustrate the benefit of using multiple complementary footprinting methods to address subtle, but possibly biologically important, differences between highly similar proteins.
Highlights
Protein footprinting coupled with mass spectrometry has become an increasingly useful strategy for the analysis of protein structure and dynamics[3,4] for larger proteins or more heterogeneous samples typically not amenable to high-resolution structural techniques such as NMR, X-ray crystallography, or cryo-EM
Two protein footprinting methods frequently used in conjunction with mass spectrometry are hydrogen−deuterium exchange (HDX)[5] and fast photochemical oxidation of proteins (FPOP).[6]
In a typical HDX-MS experiment, the protein analyte is incubated for varying lengths of time in a deuterated buffer solution, conditions under which solvent-accessible and labile hydrogen atoms on the protein (i.e., O−H, N−H, and S−H groups not involved in hydrogen bonds7) exchange, over time, with the deuterium in the buffer, increasing the mass of these regions of the protein molecule
Summary
Two protein footprinting methods frequently used in conjunction with mass spectrometry are hydrogen−deuterium exchange (HDX)[5] and fast photochemical oxidation of proteins (FPOP).[6] In a typical HDX-MS experiment, the protein analyte is incubated for varying lengths of time in a deuterated buffer solution, conditions under which solvent-accessible and labile hydrogen atoms on the protein (i.e., O−H, N−H, and S−H groups not involved in hydrogen bonds7) exchange, over time, with the deuterium in the buffer, increasing the mass of these regions of the protein molecule. The HDX reaction is quenched at low temperature and pH to minimize further forward exchange, as well as any back exchange, before the protein analyte is digested, typically with acid proteases (usually pepsin8) and the resulting peptides subjected to LC−MS/MS analysis. Rapid back exchange usually limits HDX to the study of backbone amide hydrogens,[9] this method has proved to be a key tool in the field of structural MS to study protein conformational changes as well as protein−protein and protein−ligand interactions.[5,7]
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