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Hydrogen-deuterium exchange-mass spectrometry (HDXMS) is a powerful technology to characterize conformations and conformational dynamics of proteins and protein complexes. HDXMS has been widely used in the field of therapeutics for the development of protein drugs. Although sufficient sequence coverage is critical to the success of HDXMS, it is sometimes difficult to achieve. In this study, we developed a HDXMS data analysis strategy that includes parallel post-translational modification (PTM) scanning in HDXMS analysis. Using a membrane-delimited G protein-coupled receptor (vasopressin type 2 receptor; V 2 R) and a cytosolic protein (Na + /H + exchanger regulatory factor-1; NHERF1) as examples, we demonstrate that this strategy substantially improves protein sequence coverage, especially in key structural regions likely including PTMs themselves that play important roles in protein conformational dynamics and function.

Parallel Post-Translational Modification Scanning Enhancing Hydrogen–Deuterium Exchange-Mass Spectrometry Coverage of Key Structural Regions