Organic Solvents for Enhanced Proteolysis of Stable Proteins for Hydrogen-Deuterium Exchange Mass Spectrometry.
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ABSTRACT:
SUBMITTER: Guo C
PROVIDER: S-EPMC7485609 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Hydrogen/deuterium exchange mass spectrometric (H/DXMS) methods for protein structural analysis are conventionally performed in solution. We present Tissue Deuterium Exchange Mass Spectrometry (TDXMS), a method to directly monitor deuterium uptake on tissue, as a means to better approximate the deuterium exchange behavior of proteins in their native microenvironment. Using this method, a difference in deuterium uptake behavior was observed when the same proteins were monitored in solution and on tissue. The higher maximum deuterium uptake at equilibrium for all proteins analyzed in solution suggests a more open conformation in the absence of interacting partners normally observed on tissue. We also demonstrate a difference in the deuterium uptake behavior of a few proteins across different morphological regions of the same tissue section. Modifications of the total number of hydrogens exchanged, as well as the kinetics of exchange, were both observed. These results provide information on the implication of protein interactions with partners as well as on the conformational changes related to these interactions, and illustrate the importance of examining protein deuterium exchange behavior in the presence of its specific microenvironment directly at the level of tissues.
| S-EPMC5054352 | biostudies-literature
Project description:Studies of protein dynamics, structure and interactions using hydrogen/deuterium exchange mass spectrometry (HDX-MS) have sharply increased over the past 5-10 years. The predominant technology requires fast digestion at pH 2-3 to retain deuterium label. Pepsin is used almost exclusively, but it provides relatively low efficiency under the constraints of the experiment, and a selectivity profile that renders poor coverage of intrinsically disordered regions. In this study we present nepenthesin-containing secretions of the pitcher plant Nepenthes, commonly called monkey cups, for use in HDX-MS. We show that nepenthesin is at least 1400-fold more efficient than pepsin under HDX-competent conditions, with a selectivity profile that mimics pepsin in part, but also includes efficient cleavage C-terminal to "forbidden" residues K, R, H, and P. High efficiency permits a solution-based analysis with no detectable autolysis, avoiding the complication of immobilized enzyme reactors. Relaxed selectivity promotes high coverage of disordered regions and the ability to "tune" the mass map for regions of interest. Nepenthesin-enriched secretions were applied to an analysis of protein complexes in the nonhomologous end-joining DNA repair pathway. The analysis of XRCC4 binding to the BRCT domains of Ligase IV points to secondary interactions between the disordered C-terminal tail of XRCC4 and remote regions of the BRCT domains, which could only be identified with a nepenthesin-based workflow. HDX data suggest that stalk-binding to XRCC4 primes a BRCT conformation in these remote regions to support tail interaction, an event which may be phosphoregulated. We conclude that nepenthesin is an effective alternative to pepsin for all HDX-MS applications, and especially for the analysis of structural transitions among intrinsically disordered proteins and their binding partners.
| S-EPMC3567866 | biostudies-literature
Project description:Hydrogen-deuterium exchange mass spectrometry is an important method for protein structure-function analysis. The bottom-up approach uses protein digestion to localize deuteration to higher resolution, and the essential measurement involves centroid mass determinations on a very large set of peptides. In the course of evaluating systems for various projects, we established two (HDX-MS) platforms that consisted of a FT-MS and a high-resolution QTOF mass spectrometer, each with matched front-end fluidic systems. Digests of proteins spanning a 20-110 kDa range were deuterated to equilibrium, and figures-of-merit for a typical bottom-up (HDX-MS) experiment were compared for each platform. The Orbitrap Velos identified 64% more peptides than the 5600 QTOF, with a 42% overlap between the two systems, independent of protein size. Precision in deuterium measurements using the Orbitrap marginally exceeded that of the QTOF, depending on the Orbitrap resolution setting. However, the unique nature of FT-MS data generates situations where deuteration measurements can be inaccurate, because of destructive interference arising from mismatches in elemental mass defects. This is shown through the analysis of the peptides common to both platforms, where deuteration values can be as low as 35% of the expected values, depending on FT-MS resolution, peptide length and charge state. These findings are supported by simulations of Orbitrap transients, and highlight that caution should be exercised in deriving centroid mass values from FT transients that do not support baseline separation of the full isotopic composition.
| S-EPMC3567872 | biostudies-other
Project description:Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful tool for protein structure analysis that is well suited for biotherapeutic development and characterization. Because HDX is strongly dependent on solution conditions, even small variations in temperature or pH can have a pronounced effect on the observed kinetics that can manifest in significant run-to-run variability and compromise reproducibility. Recent attention has been given to the development of internal exchange reporters (IERs), which directly monitor changes to exchange reaction conditions. However, the currently available small peptide IERs are only capable of sampling a very narrow temporal window and are understood to exhibit complex solution dependent exchange behavior. Here we demonstrate the use of imidazolium carbon acids as superior IERs for HDX-MS. These compounds exhibit predictable exchange behavior under a wide variety of reaction conditions, are highly stable, and can be readily modified to exchange over a broad temporal window. The use of these compounds as IERs for solution based HDX-MS could considerably extend the utility of the technique by allowing for more robust empirical exchange correction, thereby improving reproducibility.
| S-EPMC8106947 | biostudies-literature
Project description:Functional regulation of ligand-activated receptors is driven by alterations in the conformational dynamics of the protein upon ligand binding. Differential hydrogen/deuterium exchange (HDX) coupled with mass spectrometry has emerged as a rapid and sensitive approach for characterization of perturbations in conformational dynamics of proteins following ligand binding. While this technique is sensitive to detecting ligand interactions and alterations in receptor dynamics, it also can provide important mechanistic insights into ligand regulation. For example, HDX has been used to determine a novel mechanism of ligand activation of the nuclear receptor peroxisome proliferator activated receptor-?, perform detailed analyses of binding modes of ligands within the ligand-binding pocket of two estrogen receptor isoforms, providing insight into selectivity, and helped classify different types of estrogen receptor-? ligands by correlating their pharmacology with the way they interact with the receptor based solely on hierarchical clustering of receptor HDX signatures. Beyond small-molecule-receptor interactions, this technique has also been applied to study protein-protein complexes, such as mapping antibody-antigen interactions. In this article, we summarize the current state of the differential HDX approaches and the future outlook. We summarize how HDX analysis of protein-ligand interactions has had an impact on biology and drug discovery.
| S-EPMC3113475 | biostudies-literature
Project description:Previous structural studies of osteoprotegerin (OPG), a crucial negative regulator of bone remodeling and osteoclastogenesis, were mostly limited to the N-terminal ligand-binding domains. It is now known that the three C-terminal domains of OPG also play essential roles in its function by mediating OPG dimerization, OPG-heparan sulfate (HS) interactions, and formation of the OPG-HS-receptor activator of nuclear factor ?B ligand (RANKL) ternary complex. Employing hydrogen-deuterium exchange MS methods, here we investigated the structure of full-length OPG in complex with HS or RANKL in solution. Our data revealed two noteworthy aspects of the OPG structure. First, we found that the interconnection between the N- and C-terminal domains is much more rigid than previously thought, possibly because of hydrophobic interactions between the fourth cysteine-rich domain and the first death domain. Second, we observed that two hydrophobic clusters located in two separate C-terminal domains directly contribute to OPG dimerization, likely by forming a hydrophobic dimerization interface. Aided by site-directed mutagenesis, we further demonstrated that an intact dimerization interface is essential for the biological activity of OPG. Our study represents an important step toward deciphering the structure-function relationship of the full-length OPG protein.
| S-EPMC6231129 | biostudies-literature
Project description:Methodological improvements in both single particle cryo-electron microscopy (cryo-EM) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) mean that the two methods are being more frequently used together to tackle complex problems in structural biology. There are many benefits to this combination, including for the analysis of low-resolution density, for structural validation, in the analysis of individual proteins versus the same proteins in large complexes, studies of allostery, protein quality control during cryo-EM construct optimization, and in the study of protein movements/dynamics during function. As will be highlighted in this review, through careful considerations of potential sample and conformational heterogeneity, many joint studies have recently been demonstrated, and many future studies using this combination are anticipated.
| S-EPMC7502526 | biostudies-literature
Project description:Identification and confirmation of known as well as unknown (bio)chemical entities in ambient mass spectrometry (MS) and MS imaging (MSI) mostly involve accurate mass determination, often in combination with MS/MS or MSn work flows. To further improve structural assignment, additional molecular information is required. Here we present an ambient hydrogen/deuterium exchange (HDX) laser ablation electrospray ionization (LAESI) MS method in which, apart from the accurate mass and MS/MS data, the number of exchangeable protons in (un)known molecules is obtained. While eventually presenting ambient HDX-LAESI-MSI, samples were not preincubated with deuterated solvents, but instead HDX occurred following fusion of ablated sample material with microdroplets generated by ESI of deuterated solvents. Therefore, the degree of HDX was first studied following ablation of nondeuterated sample solutions of melamine and monosaccharides. From these experiments, it was concluded that the set-up used could provide meaningful HDX data in support of molecular structure elucidation by significantly reducing the number of structure options from a measured elemental composition. This reduction was demonstrated with an unknown accurate m/z value obtained in the analysis of an orange slice, reducing the possible number of molecular structures having the same elemental composition by 87% due to the number of H/D exchanges observed. Next, deuterated and nondeuterated MS/MS experiments showed the number of exchangeable protons in the substructures from deuterated neutral losses in the product ion spectra, confirming the compound to be arginine. Finally, the potential of ambient HDX-LAESI-MSI was demonstrated by the imaging of (secondary) plant metabolites in a Phalaenopsis petal.
| S-EPMC7053432 | biostudies-literature
Project description:Isobaric ions having the same mass-to-charge ratio cannot be separately identified by mass spectrometry (MS) alone, but this limitation can be overcome by using hydrogen-deuterium exchange (HDX) in microdroplets. Because isobaric ions may contain a varied number of exchangeable sites and different types of functional groups, each one produces a unique MS spectral pattern after droplet spray HDX without the need for MS/MS experiments or introduction of ion mobility measurements. As an example of the power of this approach, isobaric ions in urinary metabolic profiles are identified and used to distinguish between healthy individuals and those having bladder cancer.
| S-EPMC8625015 | biostudies-literature
Project description:Hydrogen deuterium exchange mass spectrometry (H/DX MS) provides a quantitative comparison of the relative rates of exchange of amide protons for solvent deuterons. In turn, the rate of amide exchange depends on a complex combination of the stability of local secondary structure, solvent accessibility, and dynamics. H/DX MS has, therefore, been widely used to probe structure and function of soluble proteins, but its application to membrane proteins was limited previously to detergent solubilized samples. The large excess of lipids from model membranes, or from membrane fractions derived from in vivo samples, presents challenges with mass spectrometry. The lipid nanodisc platform, consisting of apolipoprotein A-derived membrane scaffold proteins, provides a native like membrane environment in which to capture analyte membrane proteins with a well defined, and low, ratio of lipid to protein. Membrane proteins in lipid nanodiscs are amenable to H/DX MS, and this is expected to lead to a rapid increase in the number of membrane proteins subjected to this analysis. Here we review the few literature examples of the application of H/DX MS to membrane proteins in nanodiscs. The incremental improvements in the experimental workflow of the H/DX MS are described and potential applications of this approach to study membrane proteins are described.
| S-EPMC6480397 | biostudies-literature