Unambiguous Assignment of Intramolecular Chemical Cross-Links in Modified Mammalian Membrane Proteins by Fourier Transform-Tandem Mass Spectrometry
| Autor: | Marites J. Ayson, Richard B. Jacobsen, Petr Novák, Leavell, Joseph S. Schoeniger, William E. Haskins, Gary Kruppa, Malin M. Young |
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| Rok vydání: | 2005 |
| Předmět: |
Ions
chemistry.chemical_classification Rhodopsin Fourier Analysis Electron-capture dissociation Molecular Sequence Data Analytical chemistry Peptide Rod Cell Outer Segment Tandem mass spectrometry Dissociation (chemistry) Fourier transform spectroscopy Analytical Chemistry Crystallography symbols.namesake Fourier transform chemistry Tandem Mass Spectrometry Intramolecular force symbols Animals Cattle Amino Acid Sequence Infrared multiphoton dissociation |
| Zdroj: | Analytical Chemistry. 77:5101-5106 |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/ac040194r |
| Popis: | 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 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. |
| Databáze: | OpenAIRE |
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