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

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; V2R) 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.