Unambiguous Assignment of Intramolecular Chemical Cross-Links in Modified Mammalian Membrane Proteins by Fourier Transform-Tandem Mass Spectrometry

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Abstract
Fourier transform tandem mass spectrometry (FT-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 Rho50-86 (K66 or K67) cross-linked to Rho310-317 (C316) or Rho318-348 (K325 or K339), 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 (C322 and C323) and cross-linking of K67 (and not K66) to C316, K325, and K339. ECD spectra contained crucial c- and z-ions resulting from cleavage of the bond between K66 and K67. 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.