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

Abstract

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.

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

“…Using a standard peptide or well-established protein to quantitatively assess proteolytic efficiency is an effective way to achieve more reproducible digestion profiles among data sets and across different laboratories. 635 …”

“…These observations point out that standardization of conditions and reagents is critical for generating consistent digests so that peptides can be compared directly between sample sets. Using a standard peptide or well-established protein to quantitatively assess proteolytic efficiency is an effective way to achieve more reproducible digestion profiles among data sets and across different laboratories …”

Inclusion of deuterated glycopeptides provides increased sequence coverage in hydrogen/deuterium exchange mass spectrometry analysis of SARS‐CoV‐2 spike glycoprotein

“…13 Labeling of spike's "hinge" is a novel HDX-MS result, completely based on the coverage of deuterated glycopeptides containing glycan-modified N1134 (Figures 6 and S2). Robust labeling (24-30%) was observed in the fusion peptide (823-851), N-terminus, [19][20][21][22][23][24][25][26][27][28][29][30][31] and middle helix of the stalk (1174-1197).…”

“…22 A key step in modern HDX-MS analysis for obtaining sequence coverage is on-line proteolytic digestion 23 of the (glyco)protein of interest to generate peptides amenable to liquid chromatography (LC)/MS detection. HDX-MS sample preparation typically includes a low-pH ($2.5) quenching step immediately before proteolytic digestion, 12 limiting digestion to acid-tolerant proteases such as pepsin 24 or aspergillopepsin. 23,25 These proteases generate overlapping, mostly nonspecific 26 peptides that are nonetheless reproducible for a given protein substrate.…”

Mass Spectrometry Methods for Measuring Protein Stability

“…The MS equipment and techniques used to measure HDX are continually evolving. Key challenges include limiting the back-exchange of absorbed deuterium , and improving both the throughput and coverage of the protein digestion step. , Many excellent reviews cover the current applications and practice of HDX-MS. ,,,,,− As such, the discussion below focuses mainly on the application of HDX-MS for the assessment of protein stability. The earliest examples of such experiments focus on model proteins, such as ubiquitin and lysozyme, with the latter protein probed under conditions promoting both intact and reduced disulfide bonds .…”