Extraction of local hydrogen exchange data from HDX CAD MS measurements by deconvolution of isotopic distributions of fragment ions

Focus: Hydrogen Exchange And Covalent Modification


Hydrogen/deuterium exchange (HDX) coupled to protein fragmentation either in solution (by means of proteolysis) or in the gas phase (using collisional activation of protein ions) and followed by mass spectral measurements of deuterium content of individual fragments has become one of the major experimental tools to probe protein structure and dynamics. One difficulty, which often arises in the course of interpretation of HDX MS data, is a need to separate deuterium contribution to the observed isotopic patterns from that of naturally occurring isotopes. Another frequently encountered problem, especially when HDX in solution is followed by protein ion fragmentation in the gas phase, is a need to determine the deuterium content of an internal protein segment based on the measured isotopic distributions of overlapping fragments. While several algorithms were developed in the past several years to address the first problem, the second one did not enjoy as much attention. Here we report a new algorithm based on a maximum entropy principle, which is capable of extracting local exchange data form the isotope distribution of overlapping fragments, as well as subtracting the background due to the presence of natural isotopes and residual deuterium in exchange buffers. The new method is tested with several proteins and appears to generate stable solutions even under unfavorable circumstances, e.g., when the resolving power of a mass analyzer is not sufficient to avoid signal interference or when the isotopic distributions of individual fragments are complex and cannot be approximated with simple binomial distributions. The latter feature makes the algorithm particularly useful when the exchange in solution is correlated or semicorrelated, paving the way to precise structural characterization of non-native protein states in solution.


  1. 1.
    Kaltashov, I. A.; Eyles, S. J. Studies of Biomolecular Conformations and Conformational Dynamics by Mass Spectrometry. Mass Spectrom. Rev. 2002, 21, 37–71.CrossRefGoogle Scholar
  2. 2.
    Wales, T. E.; Engen, J. R. Hydrogen Exchange Mass Spectrometry for the Analysis of Protein Dynamics. Mass Spectrom. Rev. 2006, 25, 158–170.CrossRefGoogle Scholar
  3. 3.
    Konermann, L.; Simmons, D. A. Protein Folding Kinetics and Mechanisms Studied by Pulse-Labeling and Mass Spectrometry. Mass Spectrom. Rev. 2003, 22, 1–26.CrossRefGoogle Scholar
  4. 4.
    Simmons, D. A.; Konermann, L. Characterization of Transient Protein Folding Intermediates During Myoglobin Reconstitution by Time-Resolved Electrospray Mass Spectrometry with On-Line Isotopic Pulse Labeling. Biochemistry 2002, 41, 1906–1914.CrossRefGoogle Scholar
  5. 5.
    Zhang, Y. H.; Yan, X.; Maier, C. S.; Schimerlik, M. I.; Deinzer, M. L. Conformational Analysis of Intermediates Involved in the in Vitro Folding Pathways of Recombinant Human Macrophage Colony Stimulating Factor β by Sulfhydryl Group Trapping and Hydrogen/Deuterium Pulsed Labeling. Biochemistry 2002, 41, 15495–15504.CrossRefGoogle Scholar
  6. 6.
    Hoerner, J. K.; Xiao, H.; Kaltashov, I. A. Structural and Dynamic Characteristics of a Partially Folded State of Ubiquitin Revealed by Hydrogen Exchange Mass Spectrometry. Biochemistry 2005, 44, 11286–11294.CrossRefGoogle Scholar
  7. 7.
    Pan, J.; Wilson, D. J.; Konermann, L. Pulsed Hydrogen Exchange and Electrospray Charge-State Distribution as Complementary Probes of Protein Structure in Kinetic Experiments: Implications for Ubiquitin Folding. Biochemistry 2005, 44, 8627–8633.CrossRefGoogle Scholar
  8. 8.
    Eyles, S. J.; Kaltashov, I. A. Methods to Study Protein Dynamics and Folding by Mass Spectrometry. Methods 2004, 34, 88–99.CrossRefGoogle Scholar
  9. 9.
    He, F.; Emmett, M. R.; Hakansson, K.; Hendrickson, C. L.; Marshall, A. G. Theoretical and Experimental Prospects for Protein Identification Based Solely on Accurate Mass Measurement. J. Proteome Res. 2004, 3, 61–67.CrossRefGoogle Scholar
  10. 10.
    Hoerner, J. K.; Xiao, H.; Dobo, A.; Kaltashov, I. A. Is There Hydrogen Scrambling in the Gas Phase?: Energetic and Structural Determinants of Proton Mobility Within Protein Ions. J. Am. Chem. Soc. 2004, 126, 7709–7717.CrossRefGoogle Scholar
  11. 11.
    Krishna, M. M. G.; Hoang, L.; Lin, Y.; Englander, S. W. Hydrogen Exchange Methods to Study Protein Folding. Methods 2004, 34, 51–64.CrossRefGoogle Scholar
  12. 12.
    Xiao, H.; Hoerner, J. K.; Eyles, S. J.; Dobo, A.; Voigtman, E.; Mel’cuk, A. I.; Kaltashov, I. A. Mapping Protein Energy Landscapes with Amide Hydrogen Exchange and Mass Spectrometry: I. A Generalized Model for a Two-State Protein and Comparison with Experiment. Protein Sci. 2005, 14, 543–557.CrossRefGoogle Scholar
  13. 13.
    Zhang, Z. Q.; Guan, S. H.; Marshall, A. G. Enhancement of the Effective Resolution of Mass Spectra of High-Mass Biomolecules by Maximum Entropy-Based Deconvolution to Eliminate the Isotopic Natural Abundance Distribution. J. Am. Soc. Mass Spectrom. 1997, 8, 659–670.CrossRefGoogle Scholar
  14. 14.
    Hotchko, M.; Anand, G. S.; Komives, E. A.; Ten Eyck, L. F. Automated Extraction of Backbone Deuteration Levels from Amide H/2H Mass Spectrometry Experiments. Protein Sci. 2006, 15, 583–601.CrossRefGoogle Scholar
  15. 15.
    Chik, J. K.; Van de Graaf, J. L.; Schriemer, D. C. Quantitating the Statistical Distribution of Deuterium Incorporation to Extend the Utility of H/D Exchange MS Data. Anal. Chem. 2006, 78, 207–214.CrossRefGoogle Scholar
  16. 16.
    Del Mar, C.; Greenbaum, E. A.; Mayne, L.; Englander, S. W.; Woods, V. L., Jr. Structure and Properties of α-Synuclein and Other Amyloids Determined at the Amino Acid Level. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 15477–15482.CrossRefGoogle Scholar
  17. 17.
    Mohammad-Djafari, A.; Giovannelli, J.-F.; Demoment, G.; Idier, J. Regularization, Maximum Entropy and Probabilistic Methods in Mass Spectrometry Data Processing Problems. Int. J. Mass Spectrom. 2002, 215, 175–193.CrossRefGoogle Scholar
  18. 18.
    Buck, B.; Macaulay, V. A. Maximum Entropy in Action: A Collection of Expository Essays; Oxford University Press: New York, NY, 1991.Google Scholar
  19. 19.
    Xiao, H.; Kaltashov, I. A. Transient Structural Disorder as a Facilitator of Protein-Ligand Binding: Native H/D Exchange-Mass Spectrometry Study of Cellular Retinoic Acid Binding Protein I. J. Am. Soc. Mass Spectrom. 2005, 16, 869–879.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2006

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of Massachusetts-AmherstAmherstUSA

Personalised recommendations