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High-throughput determination of exchange rates of unmodified and PTM-containing peptides using HX-MS

ABSTRACT: Despite the widespread use of MS for hydrogen/deuterium exchange measurements, no systematic, large-scale study has been conducted to compare the observed exchange rates in unstructured peptides to the chemical exchange rates predicted from NMR-derived values. Consequently, how peptide properties and post-translational modifications influence exchange rates remain unknown. In this study, we sought to test the accuracy of predicted values by performing hydrogen exchange measurements on whole cell digests to generate an unbiased dataset of approximately 1100 peptides derived from naturally-occurring protein sequences. A remarkable 97% of observed exchange rates of peptides are within two-fold of predicted values. Using fully deuterated controls, we found that for approximately 50% of the peptides, the amino acid sequence and, consequently, the intrinsic exchange rate, are the primary contributors to back exchange. A meta-analysis of the remaining peptides physicochemical properties revealed multiple features that contribute either positively or negatively to back exchange discrepancies. Employing our workflow for comparable measurements on synthetic peptide mixtures containing post-translational modifications, and their unmodified counterparts, we show that lysine acetylation has a strong effect on the observed exchange rate, whereas serine/threonine phosphorylation does not. Our automated workflow enables high-throughput determination of exchange rates in peptide mixtures with diverse properties.

INSTRUMENT(S): Orbitrap Ascend, Q Exactive HF

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Steven A. Carr

PROVIDER: MSV000094510 | MassIVE | Wed Apr 10 12:19:00 BST 2024

REPOSITORIES: MassIVE

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