Electron capture dissociation distinguishes a single D-amino acid in a protein and probes the tertiary structure
Open Access
- 1 July 2004
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of the American Society for Mass Spectrometry
- Vol. 15 (7), 1087-1098
- https://doi.org/10.1016/j.jasms.2004.04.026
Abstract
First results are reported on the application of ECD in analysis of 2+ and 3+ ions of stereoisomers of Trp-cage (NLYIQWLKDGGPSSGRPPPS), the smallest and fastest-folding protein, which exhibits a tightly folded tertiary structure in solution. The chiral recognition based on the ratios of the abundances of z 18 and z 19 fragments in ECD of 2+ ions was excellent even for a single amino acid (Tyr) D-substitution (R chiral=8.6). The chiral effect decreased with an increase of temperature at the electrospray ion source, as well as at a higher degree of ionization, 3+ ions (R chiral=1.5). A general approach is suggested for charge localization in n+ ions by analysis of ECD mass spectra of (n+1)+ ions. Application of this approach to 3+ Trp-cage ions revealed the protonation probability order in 2+ ions: Arg16≫Gln5 >≈ N-terminus. The ECD results for native form of the 2+ ions favor the preservation of the solution-phase tertiary structure, and chiral recognition through the interaction between the charges and the neutral bond network. Conversely, ECD of 3+ ions supports the dominance of ionic hydrogen bonding which determines a different gas-phase structure than found in solution. Vibrational activation of 2+ ions indicated greater stability of the native form, but the fragmentation patterns did not provide stereoisomer differentiation, thus underlying the special position of ECD among other MS/MS fragmentation techniques. Further ECD studies should yield more structural information as well as quantitative single-amino acid D/l content measurements in proteins.Keywords
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