Digestion Optimization

The digestion conditions are optimized prior to conducting multiple H/D exchange experiments, to ensure high sequence coverage. Calculation of the differences in deuterium content between overlapping peptides is the preferred method to localize incorporated deuterium atoms [6, 22]. Variable digestion parameters include the type and bed volume of the protease columns, the transit time of the protein over the protease column, the type and concentration of denaturant [6], and inclusion of reducing reagents such as Tris(2-carboxyethyl)phos-phine hydrochloride (TCEP) [46].

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