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Hydrogen Deuterium Exchange (HDX) Mass Spectrometry (MS) is a sensitive analytical technique that provides information on protein conformation and dynamics in solution. It is commonly used in the study of protein–ligand and protein–protein interactions and more recently in the pharmaceutical industry for epitope mapping, screening drug candidates and in the comparison of biopharmaceuticals to biosimilars. HDX-MS monitors the exchange of protein backbone hydrogen atoms with deuterium in solution. Recent advancements in HDX automation and data analysis, have taken the emphasis off developing a fundamental understanding of HDX, which is still lacking. This tutorial review will cover the different mechanisms of exchange and how the exchange reaction is affected by various factors. We also explore the basis of data analysis and the difficulties that often arise in the interpretation of site-specific and segment-averaged HDX data, such as overlapping isotopic distributions and correct identification of peptides. Initial data analysis generates a list of peptides and the deuterium incorporation of each peptide at each labeling time point, i.e., a set of deuterium uptake profiles. Data interpretation and error analysis is subsequently required to ensure that deuterium uptake profiles accurately reflect conformational dynamics in solution. Finally, this review will also discuss the different ways in which HDX data can be represented and how the data can be interpreted. [ABSTRACT FROM AUTHOR]

Many important chemical and biochemical phenomena proceed on sub-second time scales before entering equilibrium. In this mini-review, we explore the history and recent advancements of time-resolved mass spectrometry (TRMS) for the characterization of millisecond time-scale chemical reactions and biochemical processes. TRMS allows for the simultaneous tracking of multiple reactants, intermediates and products with no chromophoric species required, high sensitivity and temporal resolution. The method has most recently been used for the characterization of several short-lived reaction intermediates in rapid chemical reactions. Most of the reactions that occur in living organisms are accelerated by enzymes, with pre-steady state kinetics only attainable using time-resolved methods. TRMS has been increasingly used to monitor the conversion of substrates to products and the resulting changes to the enzyme during catalytic turnover. Early events in protein folding systems have also been elucidated, along with the characterization of dynamics and transient secondary structures in intrinsically disordered proteins. In this review, we will highlight representative examples where TRMS has been applied to study these phenomena. [ABSTRACT FROM AUTHOR]

Protein unfolding intermediates are thought to play a critical role in conformational pathogenesis, acting as a ‘gateway’ to inactivation or pathogenic aggregation. Unfolding intermediates have long been studied either by populating partially-folded species at equilibrium using incresingly denaturing conditions, or by transiently populating ‘kinetic’ intermediates under fully denaturing conditions using a time-resolved approach (e.g. stopped-flow fluorescence). However, it is not clear that the folding intermediates populated under equilibrium conditions are comparable to intermediates transiently populated in kinetic experiments. In this work, we combine time-resolved electrospray (TRESI) with travelling wave Ion Mobility Spectrometry (IMS) for the first time to directly compare equilibrium and kinetic unfolding intermediates of cytochrome c. Our results show a high degree of correlation between all species populated under these substantially different regimes. [ABSTRACT FROM AUTHOR]

Here we introduce pre-equilibration kinetic size-exclusion chromatography with mass-spectrometry detection (peKSEC-MS), which is a label-free solution-based kinetic approach for characterizing non-covalent protein–small molecule interactions. In this method, a protein and a small molecule are mixed outside the column and incubated to approach equilibrium. The equilibrium mixture is then introduced into the SEC column to initiate the dissociation process by separating small molecules from the complex inside the column. A numerical model of a 1-dimensional separation was constructed to simulate mass chromatograms of the small molecule for varying rate constants of binding. [ABSTRACT FROM AUTHOR]

In this work, we introduce an integrated, electrospray mass spectrometry-coupled microfluidic chip that supports the complete workflow for ‘bottom up’ hydrogen/deuterium exchange (HDX) pulse labelling experiments. HDX pulse labelling is used to measure structural changes in proteins that occur after the initiation of a reaction, most commonly folding. In the present case, we demonstrate the device on the β-lactamase enzyme TEM-1, identifying active site changes that occur upon acylation by a covalent inhibitor and subtle changes in conformational dynamics that occur away from the active site over a period of several second after the inhibitor is bound. Our results demonstrate the power of microfluidics-enabled sub-second HDX pulse labelling as a tool for studying allostery and show some intriguing correlations with mutagenesis studies. [ABSTRACT FROM AUTHOR]