Journal
ANALYTICAL CHEMISTRY
Volume 77, Issue 16, Pages 5101-5106Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac040194r
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Fourier transform tandem mass spectrometry (Fr-MS/MS) can be used to unambiguously assign intramolecular chemical cross-links to specific amino acid residues even when two or more possible cross-linking sites are adjacent in the cross-linked protein. Bovine rhodopsin (Rho) in its dark-adapted state was intramolecularly cross-linked with lysine-cysteine (K-C) or lysine-lysine (K-K) cross-linkers to obtain interatomic distance information. Large, multiply charged, cross-linked peptide ions containing adjacent lysines, corresponding to Rho(50-86) (K-66 or K-67) cross-linked to Rho(310-317) (C-316) or Rho(318-348) (K-325 or K-339), were fragmented by collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD), and electron capture dissociation (ECD). Complementary sequence-specific information was obtained by combining cross-link assignments; however, only ECD revealed full palmitoylation of adjacent cysteines (C-322 and C-323) and cross-linking of K-67 (and not K-66) to C-316, K-325, and K-339. ECD spectra contained crucial c- and z-ions resulting from cleavage of the bond between K-66 and K-67. To our knowledge, this work also presents the first demonstration that ECD can be used to characterize S-linked fatty acid acylation on cysteines. The comprehensive fragmentation of large peptides by CID, IRMPD, and particularly ECD, in conjunction with the high resolution and mass accuracy of FT-MS/MS, is shown to be a valuable means of characterizing mammalian membrane proteins with both chemical and posttranslational modifications.
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