Changes in protein structure monitored by use of gas‐phase hydrogen/deuterium exchange

Abstract

The study of protein conformation by solution‐phase hydrogen/deuterium exchange (HDX) coupled to MS is well documented. This involves monitoring the exchange of backbone amide protons with deuterium and provides details concerning the protein's tertiary structure. However, undesired back‐exchange during post‐HDX analyses can be difficult to control. Here, gas‐phase HDX‐MS, during which labile hydrogens on amino acid side chains are exchanged in sub‐millisecond time scales, has been employed to probe changes within protein structures. Addition of the solvent 2,2,2‐trifluoroethanol to a protein in solution can affect the structure of the protein, resulting in an increase in secondary and/or tertiary structure which is detected using circular dichroism. Using a Synapt G2‐S ESI‐mass spectrometer modified to allow deuterated ammonia into the transfer ion guide (situated between the ion mobility cell and the TOF analyser), gas‐phase HDX‐MS is shown to reflect minor structural changes experienced by the proteins β‐lactoglobulin and ubiquitin, as observed by the reduction in the level of deuterium incorporation. Additionally, the use of gas‐phase HDX‐MS to distinguish between co‐populated proteins conformers within a solution is demonstrated with the disordered protein calmodulin; the gas‐phase HDX‐MS results correspond directly with complementary data obtained by use of ion mobility spectrometry‐MS.

Side-chain moieties from the N-terminal region of Aβ are Involved in an oligomer-stabilizing network of interactions

“…Gas-phase HDX-MS experiments were carried out on the oligomeric forms of these peptides in the cone-exit region of the mass spectrometer, using a setup described in more detail elsewhere [ 45 ]. Gas-phase HDX using ND 3 gas, executed at short timescales after ESI, can report on conformational differences between different structural forms of peptides and proteins [ 36 , 37 , 45 , 51 ]. In contrast to solution-phase HDX-MS, where the labeling of backbone amide hydrogens occurs across seconds to several hours, millisecond timescale gas-phase HDX-MS reports more directly on structure, with less influence from dynamics and flexibility.…”

“…Native mass spectrometry (native MS) provides such an alternative since it allows to resolve signals originating from species of different non-covalently stabilized forms that coexist in solution [ 33 ], for instance oligomers of different orders or their alternative structural forms. Inside the mass spectrometer, gaseous protein ions can be probed by a panel of gas phase techniques [ 34 ], like ion mobility (IM) [ 35 ] that additionally resolves species according to their collisional cross section (Ω, Å 2 ) or gas-phase HDX [ 36 , 37 ] which probes the involvement of side-chain protons in intra- or intermolecular H-bonding. MS-based methodology can thus also provide structural information for each of the protein species detected separately.…”

Probing the Binding Interfaces of Protein Complexes Using Gas-Phase H/D Exchange Mass Spectrometry

“…This could explain the 5 D increase in HDX of singly protonated desGly9-species relative to intact vasopressin, which in turn could be assigned to an unshielding of Arg8 (3 D), C terminus (1 D), and the charge-carrying proton (1 D). Thus, the desGly-vasopressin versus vasopressin comparison provides further evidence that gas-phase HDX-MS can report directly on structural differences between peptide/ protein ions (in terms of intramolecular interactions involving exchangeable side-chain hydrogens) provided that the charge is the same (Beeston et al, 2015;Mistarz et al, 2014;Rand et al, 2009Rand et al, , 2012.…”

“…It has emerged that a key requisite to the use of gas-phase HDX to inform on solution-phase protein conformers is to complete the labeling reaction within a few tens of milliseconds after ionization (Rand et al, 2009(Rand et al, , 2012. Gas-phase HDX performed at such millisecond timescales using ND 3 gas shortly after ESI (referred to herein as gas-phase HDX-MS) allow the selective labeling of heteroatom-bound non-amide hydrogens, and can detect and report on conformational differences between protein conformers pertinent to the solution phase (Beeston et al, 2015;Mistarz et al, 2014;Rand et al, 2009Rand et al, , 2012. The hydrogen/deuterium exchange rates of different sites in an unstructured peptide ion in the gas phase depend on the difference between the gas-phase basicity (GB) of the donor (ND 3 ) and the acceptor site (Campbell et al, 1995;Cheng and Fenselau, 1992).…”

Advances in ion mobility-mass spectrometry instrumentation and techniques for characterizing structural heterogeneity

“…132,133 Although models were developed to describe the observed exchange levels, 133,134 refined structural information from the combined IM-MS and HDX approach awaited the development of non-ergodic ion fragmentation techniques such as electron capture dissociation (ECD) 135 and electron transfer dissociation (ETD) 136 . 143 The resulting data was shown to correlate to mobility information obtained from IM-MS measurements. 137 142 Ashcroft and coworkers monitored changes in protein ion structure resulting from solution perturbations using gas-phase HDX-MS techniques.…”