Hydrogen exchange mass spectrometry (HXMS) reports changes in protein dynamics and conformations in solution by measuring the backbone amide hydrogen exchange in deuterated water. HXMS analyses have proven invaluable for elucidating unique patterns of conformational mobility that underlie phosphorylation-dependent activation in two structurally similar MAPKs, ERK1 and ERK2. While helpful in gaining an initial view of how protein motions affect enzyme activation, these studies are limited by low resolution in determining sites of deuteration. Deuteron localization at specific amides can be determined in two ways: increasing the number of pepsin-generated peptides that overlap in sequence and by implementing MS/MS sequencing of deuterated peptides free of deuteron scrambling.
State-of-the-art technology using the Waters’ Synapt G2 mass spectrometer and HX UPLC separation module provides two significant routes to circumvent these limitations for the analysis of ERK2. First, we present optimized methods for identifying more peptides with overlapping sequences. Coupling UPLC with a temperature-controlled unit allows semi-automation of sample processing and online proteolysis. Using UPLC, peptides are resolved into narrower peaks and interrogated using MSe, a new MS acquisition mode, to simultaneously fragment co-eluting ions. Novel software resolves fragment ions for peptide identification. These methods currently increase the number of identified peptides by ~3-fold on average and yield greater resolution, down to single amides in many cases. Second, we implement ETD for gas phase peptide fragmentation free of vibrational excitation, thus eliminating deuteron scrambling.
A new maximum likelihood estimation algorithm for modeling deuteration sites and rate constants allows us to gain a more accurate view of exchange at individual amides following improved HXMS analysis. Applying these methods to mitogen-activated protein kinase ERK2 affords an in-depth look at the regulatory role of protein motions in kinase activation at a level not previously possible.