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Hydrogen Deuterium Exchange (HDX) Mass Spectrometry
Elucidating protein conformation, structure and dynamics with hydrogen deuterium exchange mass spectrometry
Utilizing a HDX-MS protocol takes advantage of the labile nature of protons present on protein backbone amides, and is a powerful tool in the study of protein structure. When dissolved in solution, proteins exchange these protons with hydrogen groups present in a deuterated buffer, and protons from the protein are exchanged with deuterium. Only the protons present on the backbone amides are measured. The rate of hydrogen to deuterium exchange provides solvent accessibility data, which can be used to infer information on protein structure and conformation. Mass spectrometry can be used to measure the rate of deuterium uptake.
HDX-MS analysis can be used to obtain information on structure, protein-protein or protein-ligand interaction sites, allosteric effects, intrinsic disorder, and conformational changes induced by posttranslational modifications (PTMs). HDX-MS has the advantage of not being limited by the size of proteins or protein complexes, and it is highly sensitive, able to detect coexisting protein conformations.
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How does HDX-MS work?
All HDX-MS experiments involve deuterium labeling prior to MS analysis. The protein is incubated in a deuterium buffer, which allows for the amide hydrogens present on the protein backbone to exchange with the deuterium buffer. The most commonly used labeling approach is continuous labeling, in which a protein in its steady state is incubated in deuterium buffer continuously over different time periods, and the exchange of hydrogen to deuterium is measured as a function of time. The time period can span from seconds to hours or days. After labeling, the samples are quenched by lowering the temperature of the experiment to 0°C and the pH of the reaction to 2.5. HDX-MS experiments can be performed in either a bottom-up or intact/top-down fashion.
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On-Demand Webinar: Automated Protein Structure Analysis Using DIA Based Hydrogen Deuterium Exchange Mass Spectrometry
With Dr. David Schriemer, University of Calgary
This webinar will present an innovative approach for applying HX-MS to protein analysis in biotherapeutics development.
White paper: Hydrogen deuterium exchange mass spectrometry for the masses
HDX-MS is a powerful tool for studying protein structures, dynamics, folding, complexes and interactions. This paper will address the advantages of HDX-MS, the information it provides and the complementary role it plays with traditional techniques, as well as the recent innovations in hardware and software, and workflows that have been developed to simplify HDX-MS.
HDX-MS workflows
Identification
The most commonly used strategy for HDX-MS is to digest the proteins into peptides and analyze them using mass spectrometry. This ensures complete sequence coverage and captures region-specific information from the protein. Before hydrogen-deuterium exchange is performed, the protein is digested and analyzed in a data-dependent fashion using multiple fragmentation techniques: collision-induced dissociation [CID], higher-energy collisional dissociation [HCD] and electron transfer dissociation [ETD]).
The goal is to identify as many overlapping peptides as possible. This is done to maximize sequence coverage of the protein for identification. This process is followed by the HDX-MS experiment. Since low pH is used in HDX-MS experiments to minimize deuterium back-exchange, acidic enzymes such as pepsin are preferred for digestion. The digestion can be performed in solution or on immobilized pepsin columns, the latter being the preferred approach.
Currently available commercial platforms, such as the TRAJAN CHRONECT system, enable automated labeling and digestion. Upon digestion, the samples are desalted on a trap column and separated using reversed-phase chromatography prior to analysis by mass spectrometry.
Deuterium uptake information
The HDX-MS experiment can be performed in two ways. The first method is to collect full-scan MS to get deuterium uptake information on peptide levels to probe the protein conformation. The second method is to access higher resolution amino acid level information and thus requires acquiring peptide fragment level deuterium uptake data. Here a full-scan MS with data-dependent ETD MS2 is acquired for both unlabeled and deuterated samples. With this design, the full-scan MS experiment establishes the peptide-level deuterium incorporation value, while the higher resolution, single amino acid level deuterium incorporation value is obtained by ETD MS2. In this case, ETD is preferred over CID or HCD to avoid deuterium scrambling, a known phenomenon when energy-driven fragmentation methods are employed.
During scrambling, the protons on the peptide backbone migrate and don’t reflect the state of the peptides in solution. Studies have shown that ETD, a non-ergodic fragmentation technique, is far better suited as an activation choice due to the very low levels of hydrogen scrambling that occur during this process; therefore ETD allows for an accurate localization of incorporated deuterium at the single residue resolution level.
The alternative to bottom-up HDX-MS is intact/top-down analysis. In intact/top-down HDX-MS, proteins are introduced into the mass spectrometer after deuterium exchange without any digestion. For complex mixtures, some level of separation is performed before introducing proteins into the mass spectrometer, typically using a C4 column. Deuterium uptake may be measured at the intact level, or ETD may be employed to sequence the proteins.
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Products for HDX-MS workflow
H/D-X PAL Hydrogen Deuterium Exchange sampler system (LEAP Technologies)
The TRAJAN CHRONECT extended parallel system with syringe exchange has a standalone pepsin column chamber to enhance peptide coverage and a closed chamber for stable temperatures for all vials. A flexible 3-valve configuration in the cooling chamber allows efficient sample cleanup. Chronos software provides full editing capabilities for method customization and full integration of Thermo Scientific Xcalibur software.
Acclaim 5μm PepMap 300 μ-Precolumns Cartridge Columns
Thermo Scientific Acclaim 5μm PepMap 300 μ-Precolumns Cartridge Columns are very short microcolumns consisting of a set of disposable cartridges, and they are currently available for purchase from
Achieve exceptional peak shape and resolution for your HPLC and LC/MS applications with Thermo Scientific Hypersil GOLD HPLC columns. These endcapped, ultrapure, silica-based columns deliver significant reduction in peak tailing using generic gradients with C18 selectivity. With their excellent resolution, efficiency, and sensitivity, Hypersil GOLD columns give you confidence in the accuracy and quality of your analytical data.
The Vanquish Neo UHPLC system is the new standard in nano-, capillary-, and micro-flow liquid chromatography. Designed for researchers pursuing their next scientific breakthrough, the Vanquish Neo UHPLC system is ideal for LC-MS applications in proteomics, precision medicine, translational research, and more. Combine with Thermo Scientific PepMap Neo nano-flow LC columns for unmatched efficiency and robust performance for shallow gradients, high-throughput applications, and everything in between.
The high resolution-accurate mass (HRAM) necessary for specificity is available with Thermo Scientific Orbitrap Exploris 480 Mass Spectrometer, plus short chromatographic runs required to prevent back exchange and to allow precise measurement of deuterium incorporation. High quality Orbitrap DDA spectra in conjunction with Thermo Scientific BioPharma Finder Software ensures confident and rapid identification of all peptides
Delivering the ultimate flexibility to expand experimental scope, and with built-in intelligence, Thermo Scientific Orbitrap Eclipse Tribrid Mass Spectrometer ensures the highest data quality for HDX-MS experiments. It delivers the high resolution-accurate mass necessary for specificity with short chromatographic runs required to prevent back exchange and to allow precise measurement of deuterium incorporation. Multiple fragmentation techniques, CID, HCD and ETD are available to identify as many overlapping peptides as possible, enabling maximum sequence coverage for protein identification. Plus, it offers ultimate precision with ETD fragmentation to allow localization of deuterium exchange to the amino acid level.
Thermo Scientific BioPharma Finder software supports all HDX-MS data analysis, including peptide identification, PTM analysis and HDX-unique protection factor plots at the single residue level.
Types of information obtained using HDX-MS
A protein or protein complex can have multiple three-dimensional shapes, known as conformations. HDX-MS can provide information on the conformational differences between different states of a protein or protein complex, and can help elucidate a protein’s structured versus unstructured regions.
A protein or protein complex can undergo conformational changes to form new three-dimensional structures; this is known as protein dynamics. HDX-MS can provide information on the various short-lived, intermediate structures and the series of events that led from one state to another.
The process of biomolecule binding provides information on the interaction-interface between different subunits or ligands. The interactions may occur between a protein and another protein or between a protein and ligands such as nucleic acids, lipids, glycans and small molecules. The locations of the sites on the protein involved in the binding can be ascertained as well as how ligand binding affects protein conformation.
HDX-MS provides information on the effects of ligand binding on protein sites other than the binding site or throughout the whole protein.
The process provides information on proteins that lack a three-dimensional structure. This can be the entire protein or part of a protein that exists as flexible polypeptides or loops.
HDX-MS provides information on the regions involved in protein aggregation, conformational changes and the intermediate structures that form during aggregation.