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Hydrogen deuterium exchange (HDX) mass spectrometry (MS) is a powerful tool for studying the dynamics of higher order structure of protein-based therapeutics. The rate of hydrogen-to-deuterium exchange within the amide hydrogen on the backbone of biotherapeutics provides solvent accessibility information, and thus protein structure and conformation can be inferred.
Thermo Scientific Orbitrap-based HDX MS offers a robust method for the analysis of protein conformation, conformation dynamics, and protein-protein interactions.
Full structural characterization is critical in biopharmaceutical development where the subtle but critical local conformational changes can impact safety and efficacy.
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].
Currently available commercial platforms, such as the TRAJAN CHRONECT system, enable automated labelling and digestion.
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.
The new TRAJAN CHRONECT extended parallel system can accurately schedule fast deuterium exchange time points (approximately 15 seconds) to probe protein conformation dynamics. The TRAJAN CHRONECT autosampler includes:
A flexible 3-valve configuration in the cooling chamber allows efficient sample clean-up. The updated three valve system could set up the back-flash function to efficiently reduce the carryover and clean up the build-up on top of the column which results in very reproducible chromatography. Chronos software provides full editing capabilities for method customization and full integration of Thermo Scientific Xcalibur software.
Generate high-resolution analyses of tryptic, natural, and synthetic peptides using Acclaim PepMap HPLC columns. Due to their high loading capacity, these columns are exceptionally suitable for the analysis of low abundant peptides in complex samples.
Achieve exceptional peak shape and resolution for your LC/MS applications with 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.
Pursue the next scientific breakthrough with the best performance, productivity, and usability in a nano-, capillary-, and micro-flow UHPLC systems. The Vanquish Neo UHPLC system combines an unrivaled degree of innovation to deliver 24/7 reproducible separations of complex mixtures at maximum performance for a variety of high-sensitivity LC-MS workflow.
Deliver on your objectives by combining application flexibility with superior performance for HDX-MS to drive discovery and identification.
The high resolution-accurate mass (HRAM) necessary for specificity is available with 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
Built to meet the demands of structural biology, the Orbitrap Ascend Structural Biology Tribrid mass spectrometer adds innovations to scale up characterization of biomolecular structures, including protein-protein or protein-ligand interactions on intact level or peptide-level.
Delivering the ultimate flexibility to expand experimental scope, and with built-in intelligence, 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, UVPD 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 or UVPD 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.
Benjamin T. Walters. Scientist, Genentech
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