Membrane proteins constitute over 30% of the human proteome and represent more than 60% of drug targets, making them of critical interest in pharmaceutical discovery. Membrane proteins function within complex lipid environments that actively regulate their structure, dynamics, and activity. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) has emerged as a powerful approach for probing membrane protein dynamics in solution and native-like assemblies, including nanodiscs, SMALPs, and liposomes, paving the way to study integral membrane proteins within the context of living cells. In this perspective, we discuss HDX-MS analysis of membrane protein-lipid interactions, particularly key challenges such as lipid-induced ion suppression and chromatographic interference, and highlight recent advances in lipid removal strategies, subzero chromatography, mass spectrometry/ion mobility, and the integration of molecular dynamics simulations and artificial intelligence. We further emphasise the synergy of HDX-MS with lipidomics and native MS to better understand the interplay between lipid composition, binding stoichiometry, and structural dynamics. Together, these approaches establish an emerging multidimensional framework for understanding membrane protein-lipid interplay under physiologically relevant conditions.

While protein characterisation is critical for continuing drug discovery and development, many techniques fall short of capturing the range of conformational dynamics that underpin the targeted protein activity. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful tool for investigation of protein structural dynamics, particularly for membrane proteins in different mimetic environments. This measurement of intrinsic protein behaviour is invaluable in outlining pathogenic protein dynamics, protein-ligand profiles, and druggable protein centres that are often inaccessible in other structural techniques. This minireview will cover how recent advances have been applied to HDX-MS of membrane proteins and peptides widening the use of HDX for drug discovery.

Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is a powerful tool for studying protein structure and dynamics. As a bottom-up LC-MS technique, its success largely depends on peptide identifications made by peptide mapping prior to HDX measurements. We previously demonstrated that combining peptide mapping results from complementary single- and multipass cyclic ion mobility-mass spectrometry (cIM-MS) experiments, an approach we term "multi-sequence" cIM-MS, can enhance HDX-MS by increasing peptide identifications. However, this approach required labor-intensive, manual handling of the acquired data, including lengthy optimization of drift time (DT) versus DT full width half maximum (FWHM) trendlines during peak detection processing to combat cyclic wrap-around effects. Here, we present MultiPassMerger, an open-source software tool that automates the processing, merging, and filtering of single- and multipass cIM-MS peptide mapping data. MultiPassMerger was validated through re-analysis of several model proteins. Its automated capabilities enabled better optimization of DT versus DT FWHM trendlines on a protein-specific basis, enhancing peptide identification relative to manually optimized trendlines. Beyond automating our previous approach, MultiPassMerger also introduces a novel "multi-trendline" processing method, involving iterative processing using multiple trendlines and merging of results to better sample ions across the DT versus DT FWHM distribution. Using MultiPassMerger with both multi-sequence and multi-trendline strategies increased peptide identifications up to 392% relative to SYNAPT G2-Si with linear ion mobility and 102% relative to use of single-pass cIM-MS alone. Consequently, MultiPassMerger can enhance peptide mapping and makes this approach practical and more accessible to the wider HDX-MS community. MultiPassMerger is available as a downloadable Windows executable at https://politislab.uk/multipassmerger.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a powerful technique for studying protein structural dynamics. A critical step in the HDX-MS workflow is generating a peptide map from nondeuterated samples, which serves as the reference for identifying and monitoring peptides in subsequent deuterium-labeled experiments. Maximizing peptide identifications improves sequence coverage and redundancy, enhancing the information content and spatial resolution of the HDX-MS data. However, peptide identification is often limited by suboptimal peptide separation/fragmentation. In other proteomic workflows, longer liquid chromatography (LC) gradients are commonly used to improve the peptide identification by increasing resolution. However, in HDX-MS workflows, such gradients are generally incompatible due to time constraints imposed by deuterium/hydrogen back-exchange. To address this, we introduce a flexible workflow that uses long-gradients during initial peptide mapping, followed by retention time (RT) interpolation for application in subsequent short-gradient HDX-MS. By performing both long- and short-gradient peptide mapping, we used shared peptides to generate a regression model that predicts short-gradient RTs for all peptides identified in the long-gradient experiment. This enables the use of the richer peptide maps provided by long-gradient chromatography without compromising the deuterium retention. The method is implemented by RTinterpolator, a freely available R script compatible with widely used HDX analysis platforms that rely on reference RT values for peptide monitoring in deuterium-labeled data. By providing predicted RTs aligned to short gradients, RTinterpolator offers a practical, accessible, and instrument-independent way of increasing sequence coverage and redundancy in HDX-MS experiments, particularly for large or complex proteins susceptible to the limitations of short-gradient chromatography.

Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful tool to probe protein dynamics. As a bottom-up technique, HDX-MS provides information at peptide-level resolution, allowing structural localization of dynamic changes. Consequently, the HDX-MS data quality is largely determined by the number of peptides that are identified and monitored after deuteration. Integration of ion mobility (IM) into HDX-MS workflows has been shown to increase the data quality by providing an orthogonal mode of peptide ion separation in the gas phase. This is of critical importance for challenging targets such as integral membrane proteins (IMPs), which often suffer from low sequence coverage or redundancy in HDX-MS analyses. The increasing complexity of samples being investigated by HDX-MS, such as membrane mimetic reconstituted and in vivo IMPs, has generated need for instrumentation with greater resolving power. Recently, Giles et al. developed cyclic ion mobility (cIM), an IM device with racetrack geometry that enables scalable, multipass IM separations. Using one-pass and multipass cIM routines, we use the recently commercialized SELECT SERIES Cyclic IM spectrometer for HDX-MS analyses of four detergent solubilized IMP samples and report its enhanced performance. Furthermore, we develop a novel processing strategy capable of better handling multipass cIM data. Interestingly, use of one-pass and multipass cIM routines produced unique peptide populations, with their combined peptide output being 31 to 222% higher than previous generation SYNAPT G2-Si instrumentation. Thus, we propose a novel HDX-MS workflow with integrated cIM that has the potential to enable the analysis of more complex systems with greater accuracy and speed.

AbstractG Protein Coupled Receptors (GPCRs) constitute the largest family of signalling proteins responsible for translating extracellular stimuli into intracellular functions. They play crucial roles in numerous physiological processes and are major targets for drug discovery. Dysregulation of GPCRs is implicated in various diseases, making understanding their structural dynamics critical for therapeutic development. Here, we use Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) to explore the structural dynamics of the turkey β1-adrenergic receptor (tβ1AR) bound with nine different ligands, including agonists, partial agonists, and antagonists. We find that these ligands induce distinct dynamic patterns across the receptor, which can be grouped by compound modality. Notably, full agonist binding destabilises the intracellular loop 1 (ICL1), while antagonist binding stabilises it, highlighting ICL1’s role in G protein recruitment. Our findings indicate that the conserved L72 residue in ICL1 is crucial for maintaining receptor structural integrity and stabilising the GDP-bound state. Overall, our results provide a platform for determining drug modality and highlight how HDX-MS can be used to dissect receptor ligand interaction properties and GPCR mechanism.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has evolved as an essential technique in structural proteomics. The use of ion mobility separation (IMS) coupled to HDX-MS has increased the applicability of the technique to more complex systems and has been shown to improve data quality and robustness. The first step when running any HDX-MS workflow is to confirm the sequence and retention time of the peptides resulting from the proteolytic digestion of the nondeuterated protein. Here, we optimized the collision energy ramp of HDMSE experiments for membrane proteins using a Waters SELECT SERIES cIMS-QTOF system following an HDX workflow using Phosphorylase B, XylE transporter, and Smoothened receptor (SMO) as model systems. Although collision energy (CE) ramp 10-50 eV gave the highest amount of positive identified peptides when using Phosphorylase B, XylE, and SMO, results suggest optimal CE ramps are protein specific, and different ramps can produce a unique set of peptides. We recommend cIMS users use different CE ramps in their HDMSE experiments and pool the results to ensure maximum peptide identifications. The results show how selecting an appropriate CE ramp can change the sequence coverage of proteins ranging from 4 to 94%.

SARS-CoV-2 spike glycoprotein mediates receptor binding and subsequent membrane fusion. It exists in a range of conformations, including a closed state unable to bind the ACE2 receptor, and an open state that does so but displays more exposed antigenic surface. Spikes of variants of concern (VOCs) acquired amino acid changes linked to increased virulence and immune evasion. Here, using HDX-MS, we identified changes in spike dynamics that we associate with the transition from closed to open conformations, to ACE2 binding, and to specific mutations in VOCs. We show that the RBD-associated subdomain plays a role in spike opening, whereas the NTD acts as a hotspot of conformational divergence of VOC spikes driving immune evasion. Alpha, beta and delta spikes assume predominantly open conformations and ACE2 binding increases the dynamics of their core helices, priming spikes for fusion. Conversely, substitutions in omicron spike lead to predominantly closed conformations, presumably enabling it to escape antibodies. At the same time, its core helices show characteristics of being pre-primed for fusion even in the absence of ACE2. These data inform on SARS-CoV-2 evolution and omicron variant emergence.

Lipid interactions modulate the function, folding, structure, and organization of membrane proteins. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has emerged as a useful tool to understand the structural dynamics of these proteins within lipid environments. Lipids, however, have proven problematic for HDX-MS analysis of membrane-embedded proteins, due to their presence impairing proteolytic digestion, causing liquid chromatography column fouling, ion suppression, and/or mass spectral overlap. Here, we describe the integration of a chromatographic phospholipid trap column into the HDX-MS apparatus to enable online sample delipidation prior to protease digestion of deuterium labeled protein-lipid assemblies. We demonstrate the utility of this method on membrane scaffold protein lipid nanodisc – both empty and loaded with the ~115 kDa transmembrane protein AcrB – proving efficient and automated phospholipid capture with minimal D-to-H back-exchange, peptide carry-over, and with minimal protein loss. Our results provide insights into the efficiency of phospholipid capture by ZrO2-coated and TiO2 beads, and describe how solution conditions can be optimized to maximize the performance of our online, but also the existing offline, delipidation workflows for HDX-MS. We envision that this HDX-MS method will significantly ease membrane protein analysis, allowing to better interrogate their dynamics in artificial lipid bilayers or even cell membranes.

A yet unresolved challenge in structural biology is to quantify the conformational states of proteins underpinning function. This challenge is particularly acute for membrane proteins owing to the difficulties in stabilizing them for in vitro studies. To address this challenge, we present an integrative strategy that combines hydrogen deuterium exchange-mass spectrometry (HDX-MS) with ensemble modeling. We benchmark our strategy on wild-type and mutant conformers of XylE, a prototypical member of the ubiquitous Major Facilitator Superfamily (MFS) of transporters. Next, we apply our strategy to quantify conformational ensembles of XylE embedded in different lipid environments. Further application of our integrative strategy to substrate-bound and inhibitor-bound ensembles allowed us to unravel protein-ligand interactions contributing to the alternating access mechanism of secondary transport in atomistic detail. Overall, our study highlights the potential of integrative HDX-MS modeling to capture, accurately quantify, and subsequently visualize co-populated states of membrane proteins in association with mutations and diverse substrates and inhibitors.

Integral membrane proteins (IMPs) perform a range of diverse functions and their dysfunction underlies numerous pathological conditions. Consequently, IMPs constitute most drug targets, and the elucidation of their mechanism of action has become an intense field of research. Historically, IMP studies have relied on their extraction from membranes using detergents, which have the potential to perturbate their structure and dynamics. To circumnavigate this issue, an array of membrane mimetics has been developed that aim to reconstitute IMPs into native-like lipid environments that more accurately represent the biological membrane. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has emerged as a versatile tool for probing protein dynamics in solution. The continued development of HDX-MS methodology has allowed practitioners to investigate IMPs using increasingly native-like membrane mimetics, and even pushing the study of IMPs into the in vivo cellular environment. Consequently, HDX-MS has come of age and is playing an ever-increasingly important role in the IMP structural biologist toolkit. In the present mini-review, we discuss the evolution of membrane mimetics in the HDX-MS context, focusing on seminal publications and recent innovations that have led to this point. We also discuss state-of-the-art methodological and instrumental advancements that are likely to play a significant role in the generation of high-quality HDX-MS data of IMPs in the future.

G-protein-coupled receptors signal through cognate G proteins. Despite the widespread importance of these receptors, their regulatory mechanisms for G-protein selectivity are not fully understood. Here we present a native mass spectrometry-based approach to interrogate both biased signalling and allosteric modulation of the β 1 -adrenergic receptor in response to various ligands. By simultaneously capturing the effects of ligand binding and receptor coupling to different G proteins, we probed the relative importance of specific interactions with the receptor through systematic changes in 14 ligands, including isoprenaline derivatives, full and partial agonists, and antagonists. We observed enhanced dynamics of the intracellular loop 3 in the presence of isoprenaline, which is capable of acting as a biased agonist. We also show here that endogenous zinc ions augment the binding in receptor–G s complexes and propose a zinc ion-binding hotspot at the TM5/TM6 intracellular interface of the receptor–G s complex. Further interrogation led us to propose a mechanism in which zinc ions facilitate a structural transition of the intermediate complex towards the stable state.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the target for neutralizing antibodies elicited following both infection and vaccination. While extensive research has shown that the receptor binding domain (RBD) and, to a lesser extent, the N-terminal domain (NTD) are the predominant targets for neutralizing antibodies, identification of neutralizing epitopes beyond these regions is important for informing vaccine development and understanding antibody-mediated immune escape. Here, we identify a class of broadly neutralizing antibodies that bind an epitope on the spike subdomain 1 (SD1) and that have arisen from infection or vaccination. Using cryo-electron microscopy (cryo-EM) and hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS), we show that SD1-specific antibody P008_60 binds an epitope that is not accessible within the canonical prefusion states of the SARS-CoV-2 spike, suggesting a transient conformation of the viral glycoprotein that is vulnerable to neutralization.

The effect of temperature on the stability of proteins is well explored above 298 K, but harder to track experimentally below 273 K. Variable-temperature ion mobility mass spectrometry (VT IM-MS) allows us to measure the structure of molecules at sub-ambient temperatures. Here we monitor conformational changes that occur to two isotypes of monoclonal antibodies (mAbs) on cooling by measuring their collision cross sections (CCS) at discrete drift gas temperatures from 295 to 160 K. The CCS at 250 K is larger than predicted from collisional theory and experimental data at 295 K. This restructure is attributed to change in the strength of stabilizing intermolecular interactions. Below 250 K the CCS of the mAbs increases in line with prediction implying no rearrangement. Comparing data from isotypes suggest disulfide bridging influences thermal structural rearrangement. These findings indicate that in vacuo deep-freezing minimizes denaturation and maintains the native fold and VT IM-MS measurements at sub ambient temperatures provide new insights to the phenomenon of cold denaturation.

A yet unresolved challenge in structural biology is to quantify conformational states of proteins underpinning function. This challenge is particularly acute for membrane proteins owing to the difficulties in stabilising them for i n vitro studies. To address this challenge, we present here an integrative strategy that combines hydrogen-deuterium exchange mass spectrometry (HDX-MS) with ensemble modelling. We benchmark our strategy on wild type and mutant conformers of XylE, a prototypical member of the ubiquitous Major Facilitator Superfamily (MFS) of transporters. Next, we apply our strategy to quantify conformational ensembles of XylE embedded in different lipid environments and identify key lipid contacts that modulate protein conformations. Further application of our integrative strategy to substrate-bound and inhibitor-bound ensembles, allowed us to unravel protein-ligand interactions contributing to the alternating access mechanism of secondary transport in atomistic detail. Overall, our study highlights the potential of integrative HDX-MS modelling to capture, accurately quantify and subsequently visualise co-populated states of membrane proteins in association with mutations and diverse substrates and inhibitors. For Table of Content Only

Transporters cycle through large structural changes to translocate molecules across biological membranes. The temporal relationships between these changes and function, and the molecular properties setting their rates, determine transport efficiency-yet remain mostly unknown. Using single-molecule fluorescence microscopy, we compare the timing of conformational transitions and substrate uptake in the elevator-type transporter GltPh We show that the elevator-like movements of the substrate-loaded transport domain across membranes and substrate release are kinetically heterogeneous, with rates varying by orders of magnitude between individual molecules. Mutations increasing the frequency of elevator transitions and reducing substrate affinity diminish transport rate heterogeneities and boost transport efficiency. Hydrogen deuterium exchange coupled to mass spectrometry reveals destabilization of secondary structure around the substrate-binding site, suggesting that increased local dynamics leads to faster rates of global conformational changes and confers gain-of-function properties that set transport rates.

AbstractSummaryHydrogen deuterium exchange mass spectrometry (HDX-MS) is becoming increasing routine for monitoring changes in the structural dynamics of proteins. Differential HDX-MS allows comparison of protein states, such as in the absence or presence of a ligand. This can be used to attribute changes in conformation to binding events, allowing the mapping of entire conformational networks. As such, the number of necessary cross-state comparisons quickly increases as additional states are introduced to the system of study. There are currently very few software packages available that offer quick and informative comparison of HDX-MS datasets and even fewer which offer statistical analysis and advanced visualization. Following the feedback from our original software Deuteros, we present Deuteros 2.0 which has been redesigned from the ground up to fulfill a greater role in the HDX-MS analysis pipeline. Deuteros 2.0 features a repertoire of facilities for back exchange correction, data summarization, peptide-level statistical analysis and advanced data plotting features.Availability and implementationDeuteros 2.0 can be downloaded for both Windows and MacOS from https://github.com/andymlau/Deuteros_2.0 under the Apache 2.0 license.

The effect of temperature on the stability of proteins is well explored for high temperatures, but harder to track below the freezing point of water. This challenge is met with the use of variable temperature ion mobility mass spectrometry (VT IM-MS), which allows the structure of isolated, solvent free molecules to be measured at sub ambient temperatures in the form of their collision cross section (CCS). Here we monitor conformational changes that occur to two isotypes of monoclonal antibodies over a temperature range from 295 to 165 K. For each we observe a large increase in the magnitude of the CCS at 250K (-20 °C) substantially above that predicted. This loss of structure in the absence of bulk solvent is attributed to a change in the strength of stabilizing intermolecular interactions, causing rearrangement. At 190 K (-80 °C) the CCS distribution narrows which we attribute to better resolution. These findings indicate that in vacuo deep-freezing minimizes denaturation and maintains the gas phase native fold supporting this practice in vitro. Comparing the data for each isotype suggests that the disulfide bridging influences thermal structural rearrangement and taken together we show that this method provides unique insights to the phenomenon of cold denaturation.

Native mass spectrometry (MS) allows the interrogation of structural aspects of macromolecules in the gas phase, under the premise of having initially maintained their solution-phase non-covalent interactions intact. In the more than 25 years since the first reports, the utility of native MS has become well established in the structural biology community. The experimental and technological advances during this time have been rapid, resulting in dramatic increases in sensitivity, mass range, resolution, and complexity of possible experiments. As experimental methods are improved, there have been accompanying developments in computational approaches for analysing and exploiting the profusion of MS data in a structural and biophysical context. Here, based on discussions within the EU COST Action BM1403 on Native MS and Related Methods for Structural Biology with broad participation from Europe and North America, we consider the computational strategies currently being employed by the community, aspects of best practice, and the challenges that remain to be addressed.