H/D Exchange Centroid Monitoring is Insufficient to Show Differences in the Behavior of Protein States

  • Jun Zhang
  • Pradeep Ramachandran
  • Rajiv Kumar
  • Michael L. Gross
Application Note


Differential hydrogen/deuterium exchange (H/DX) coupled with mass spectrometry (H/DX-MS) offers a rapid and sensitive characterization of changes in proteins following perturbations induced by changes in folding, ligand binding, oligomerization, and modification. The characterization of H/DX rates by software tools and automated data processing often relies on the centroid mass calculation and, thereby, the deuterium distribution in the mass spectra is neglected. Here we present an example demonstrating the clear limitation of using only a centroid approach to characterize the H/DX rate, in which the change in protein is not reflected as the difference in deuterium uptake based on centroid calculation.

Key words

Hydrogen/deuterium exchange Mass spectrometry Centroid of D distribution DREAM protein EX1 and EX2 kinetics 



The authors thank Bruce Pascal from Professor Patrick R. Griffin’s lab at Scripps Florida for help with HDX data analysis and helpful discussion. The work was supported by grants from the National Institute of General Medical Sciences (8 P41 GM103422-35) of the NIH.

Supplementary material

13361_2012_555_MOESM1_ESM.doc (72 kb)
ESM 1(DOC 71 kb)


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Copyright information

© American Society for Mass Spectrometry 2013

Authors and Affiliations

  • Jun Zhang
    • 1
  • Pradeep Ramachandran
    • 2
    • 3
  • Rajiv Kumar
    • 2
    • 3
  • Michael L. Gross
    • 1
  1. 1.Department of ChemistryWashington University in St. LouisSt. LouisUSA
  2. 2.Division of Nephrology and Hypertension, Department of MedicineMayo ClinicRochesterUSA
  3. 3.Department of Biochemistry and Molecular BiologyMayo ClinicRochesterUSA

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