Unambiguous assignment of intramolecular chemical cross-links in modified mammalian membrane proteins by Fourier transform-tandem mass spectrometry.
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.
Novak
Petr
P
Sandia National Laboratories, Livermore, CA 94551, USA.
Haskins
William E
WE
Ayson
Marites J
MJ
Jacobsen
Richard B
RB
Schoeniger
Joseph S
JS
Leavell
Michael D
MD
Young
Malin M
MM
Kruppa
Gary H
GH
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D005583:Fourier Analysis; D007477:Ions / Q000737:chemistry; D008969:Molecular Sequence Data; D012243:Rhodopsin / Q000032:analysis* / Q000737:chemistry*; D012374:Rod Cell Outer Segment / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*
D007477:Ions; D012243:Rhodopsin