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.
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.
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.
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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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
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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.
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.