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Rapid Assessment of Pepsin Column Activity for Reliable HDX-MS Studies

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posted on 13.05.2021, 22:43 by Clint Vorauer, Michael S. Wrigley, Juan P. Rincon Pabon, Michael J. Watson, Charlie C. Mundorff, David D. Weis, Miklos Guttman
Hydrogen/deuterium exchange with mass spectrometry (HDX-MS) is a widely used technique to probe protein structural dynamics, track conformational changes, and map protein–protein interactions. Most HDX-MS studies employ a bottom-up approach utilizing the acid active protease pepsin to digest the protein of interest, often utilizing immobilized protease in a column format. The extent of proteolytic cleavage will greatly influence data quality and presents a major source of variation in HDX-MS studies. Here, we present a simple cocktail of commonly available peptides that are substrates of pepsin and can serve as a rapid check of pepsin column activity. The peptide-based assay requires no system modifications and provides an immediate readout to check and benchmark pepsin activity across different HDX-MS platforms.