Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

Abstract

Solution-phase hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) is a widespread tool for structural analysis across academia and the biopharmaceutical industry. By monitoring the exchangeability of backbone amide protons, HDX-MS can reveal information about higher-order structure and dynamics throughout a protein, can track protein folding pathways, map interaction sites, and assess conformational states of protein samples. The combination of the versatility of the hydrogen/deuterium exchange reaction with the sensitivity of mass spectrometry has enabled the study of extremely challenging protein systems, some of which cannot be suitably studied using other techniques. Improvements over the past three decades have continually increased throughput, robustness, and expanded the limits of what is feasible for HDX-MS investigations. To provide an overview for researchers seeking to utilize and derive the most from HDX-MS for protein structural analysis, we summarize the fundamental principles, basic methodology, strengths and weaknesses, and the established applications of HDX-MS while highlighting new developments and applications.

Interpreting hydrogen-deuterium exchange experiments with molecular simulations: Tutorials and applications of the HDXer ensemble reweighting software [Article v1.0]

“…The exchange reaction itself, which is governed by an intrinsic chemical exchange rate constant k int , is only possible from the amide O state. An examination of the relationship between these rate constants (Equation 1) reveals that the experimentally-observed overall rate of exchange, k HX can be considered to occupy one of two alternative kinetic regimes, known as EX1 and EX2 (Equation 2) [5,31,32].…”

“…Consequently, the experimental data used to interpret via predictive models should be carefully classified according to EX1 and EX2 behavior so that the slower exchanging amides may be excluded. Fortunately, a simple analysis of the peptide mass envelopes obtained in HDX-MS experiments often allows classification of individual peptides into EX1, EX2, or mixed EX1/EX2 exchangers for this purpose [5,31,32].…”

“…In particular, experiments that couple HDX to mass spectrometry measurements of the deuterium uptake (HDX-MS) are now widely used for analyzing the dynamics of proteins in solution. HDX-MS offers particular advantages over methods such as NMR-based exchange measurements for large proteins, complexes, and for small sample amounts [3][4][5].…”

Mass Spectrometry-Based Structural Proteomics for Metal Ion/Protein Binding Studies

“…HDX is an example of reversible labeling. Here, the protein is studied in D 2 O buffer, allowing solvent-accessible backbone amide hydrogens to exchange with deuterium [ 14 ]. Upon quench of the HDX (by bringing the solution’s pH to ~2.5), the rate reaches its minimum; thus, the deuterium is largely preserved during the analysis to follow [ 15 ].…”

Simulation of energy-resolved mass spectrometry distributions from surface-induced dissociation

“…An assortment of different techniques can be used to measure various types of structural information (1-5). For example, ion mobility (IM) can provide information on size and shape (6)(7)(8), chemical cross-linking (XL) can provide information on residue distances and contacts (9)(10)(11), and covalent labeling can provide information on solvent accessibility and flexibility of residues (12)(13)(14)(15)(16). The resulting structural information can then be used to better understand the roles of the specific proteins in biological processes.…”