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Updated: Aug 11, 2026

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics
Published on: April 17, 2017
Extensive deuterium back-exchange in certain immobilized pepsin columns used for H/D exchange mass spectrometry
Yan Wu1, Suma Kaveti, John R Engen
1Department of Chemistry, University of New Mexico, Albuquerque, New Mexico 87131-0001, USA.
Abstract:
Pepsin digestion prior to mass analysis increases the spatial resolution of hydrogen exchange mass spectrometry experiments. Online digestion with immobilized pepsin is advantageous for several reasons including better digestion efficiency. We have found that certain immobilized pepsin columns cause substantial deuterium back-exchange, rendering the data unusable. When pepsin immobilized on a POROS support was used for online digestion, back-exchange was within the expected range and was similar to the back-exchange of deuterated peptides produced by in-solution pepsin digestion. However, when pepsin immobilized onto selected polystyrene-divinylbenzene supports was used for online digestion with the same system, deuterium loss was extremely high. The effect seems linked to the properties of the solid support used to conjugate the pepsin.
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