Identification and characterization of EX1 kinetics in H/D exchange mass spectrometry by peak width analysis
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Proteins that undergo cooperative unfolding events display EX1 kinetic signatures in hydrogen exchange mass spectra. The hallmark bimodal isotope pattern observed for EX1 kinetics is distinct from the binomial isotope pattern for uncorrelated exchange (EX2), the normal exchange regime for folded proteins. Detection and characterization of EX1 kinetics is simple when the cooperative unit is large enough that the isotopic envelopes in the bimodal pattern are resolved in the m/z scale but become complicated in cases where the unit is small or there is a mixture of EX1 and EX2 kinetics. Here we describe a data interpretation method involving peak width analysis that makes characterization of EX1 kinetics simple and rapid. The theoretical basis for EX1 and EX2 isotopic signatures and the effects each have on peak width are described. Modeling of EX2 widening and analysis of empirical data for proteins and peptides containing purely EX2 kinetics showed that the amount of widening attributable to stochastic forward- and back exchange in a typical experiment is small and can be quantified. Proteins and peptides with both obvious and less obvious EX1 kinetics were analyzed with the peak width method. Such analyses provide the half-life for the cooperative unfolding event and the relative number of residues involved. Automated analysis of peak width was performed with custom Excel macros and the DEX software package. Peak width analysis is robust, capable of automation, and provides quick interpretation of the key information contained in EX1 kinetic events.
- 2.Engen, J. R.; Smith, D. L. Investigating the higher order structure of proteins: Hydrogen exchange, proteolytic fragmentation and mass spectrometry. Methods Mol. Biol. 2000, 146, 95–112.Google Scholar
- 6.Sivaraman, T.; Robertson, A. D. Kinetics of conformational fluctuations by EX1 hydrogen exchange in native proteins. Methods Mol. Biol. 2001, 168, 193–214.Google Scholar
- 17.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 2-state protein and comparison with experiment. Protein Sci. 2005, 14, 543–557.CrossRefGoogle Scholar
- 19.Weis, D. D.; Kjellen, P.; Sefton, B. M.; Engen, J. R. Altered dynamics in Lck SH3 upon binding to the LBD1 domain of Herpesvirus saimiri tip; unpublished.Google Scholar
- 23.Fu, C. Y.; Prevelige, P. E. Jr. Dynamic motions of free and bound O29 scaffolding protein identified by hydrogen deuterium exchange mass spectrometry. Protein Sci 2006; in pressGoogle Scholar