title: Electron capture dissociation distinguishes a single D-amino acid in a protein and probes the tertiary structure.
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: Arg(16) >> Gln(5) > approximately 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.
authors.affiliation: Laboratory for Biological and Medical Mass Spectrometry, Uppsala University, Uppsala, Sweden.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Adams
authors.forename: Frank
authors.initials: F
authors.lastname: Kjeldsen
authors.forename: Roman A
authors.initials: RA
authors.lastname: Zubarev
authors.forename: Bogdan A
authors.initials: BA
authors.lastname: Budnik
authors.forename: Kim F
authors.initials: KF
authors.lastname: Haselmann
mesh_terms: D000596:Amino Acids / Q000032:analysis* / Q000737:chemistry*; D003461:Crystallography / Q000379:methods*; D004583:Electrons; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000596:Amino Acids