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Updated: Jul 19, 2026

A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes
Published on: May 4, 2020
Amide proton back-exchange in deuterated peptides: applications to MS and NMR analyses
Lianmei Feng1, Ron Orlando, James H Prestegard
1Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, USA.
Abstract:
Deuterium for hydrogen exchange at amide sites in proteins is a well-established means of probing the stability of certain proteins and the effects of interactions with ligands and other proteins. When deuterium content is analyzed by mass spectrometry (MS) of digested peptides, corrections frequently need to be made for back-exchange that occurs during digestion, separation, and analysis. The back-exchange process is actually complex and deserving of analysis in a sequence-specific manner. Here an analysis of back-exchange in the decapeptide, angiotensin I, and a hexapeptide derived by digestion of a 15N-labeled carbohydrate-binding protein, galectin-3, is presented. Nuclear magnetic resonance (NMR) data are used to study back-exchange at specific sites in typical solvents used for separation and analysis, and the derived rates are found to be predictable using methods established for aqueous solvents. The predictability provides potentially new means of improving site specificity of MS analysis and new means of assigning NMR resonances for deuterium content analysis in peptides.
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