![]() ![]() Here a full-scan MS with data-dependent ETD MS2 is acquired for both unlabeled and deuterated samples. ![]() The second method is to access higher resolution amino acid level information and thus requires acquiring peptide fragment level deuterium uptake data. The first method is to collect full-scan MS to get deuterium uptake information on peptide levels to probe the protein conformation. The HDX-MS experiment can be performed in two ways. Upon digestion, the samples are desalted on a trap column and separated using reversed-phase chromatography prior to analysis by mass spectrometry. The digestion can be performed in solution or on immobilized pepsin columns, the latter being the preferred approach.Ĭurrently available commercial platforms, such as the H/D-X PAL Hydrogen Deuterium Exchange sampler system (LEAP Technologies), enable automated labeling and digestion. Since low pH is used in HDX-MS experiments to minimize deuterium back-exchange, acidic enzymes such as pepsin are preferred for digestion. This process is followed by the HDX-MS experiment. This is done to maximize sequence coverage of the protein for identification. ![]() The goal is to identify as many overlapping peptides as possible. Before hydrogen-deuterium exchange is performed, the protein is digested and analyzed in a data-dependent fashion using multiple fragmentation techniques: collision-induced dissociation, higher-energy collisional dissociation and electron transfer dissociation ). This ensures complete sequence coverage and captures region-specific information from the protein. The most commonly used strategy for HDX-MS is to digest the proteins into peptides and analyze them using mass spectrometry. ![]()
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