Hydrogen exchange mass spectrometry workflow

In a standard hydrogen exchange experiment (see Section 2.3.4 for variations in experimental workflow), the sample is deuterated for a predetermined time (milliseconds and longer times), and the reaction is slowed down several orders of magnitude by the addition of an acidic quench solution. Quenched reactions can be subjected to direct mass spectrometry analysis or immediately flash frozen in liquid nitrogen and stored at -80°C in multiple aliquots for future analysis. These flash-frozen samples need to be thawed prior to mass spectrometry. It is important to note that freeze-thaw cycles contribute to signal loss due to back-exchange (this is described further in Section 2.3.8), which... 

Figure 10.2 Schematic representation of the experimental workflow used in mass spectrometry-based experiments to determine the pK of histidine residues in proteins. The first step of the protocol involves incubation of the protein in a series of D,0 buffers at different pH values for at least 2 days before the hydrogen exchange reactions are quenched. The protein samples from each hydrogen exchange reaction are then digested with proteolytic enzyme(s), and the proteolytic fragments are analyzed by LC-TSI-MS to determine the deuterium content cjf histidine-ccjntaining peptides...

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