Localization of O-glycosylation sites in peptides by electron capture dissociation in a Fourier transform mass spectrometer.
The novel technique electron capture dissociation (ECD) of electrospray generated [M + nH]n+ polypeptide cations produces rapid cleavage of the backbone NH-Ca bond to form c and z ions (in the modified notation of Roepstorff and Fohlman). The potential of the Fourier transform mass spectrometry equipped with ECD in structure analysis of O-glycosylated peptides in the 3 kDa range has been investigated. Totally, 85% of the available interresidue bonds were cleaved in five glycopeptides; more stable c ions accounted for 62% of the observed fragmentation. The c series provided direct evidence on the glycosylation sites in every case studied, with no glycan (GalNAc and dimannose) losses observed from these species. Less stable z ions supported the glycan site assignment, with minor glycan detachments. These losses, as well as the observed formation of even-electron z ions, are attributed to radical-site-initiated reactions. In favorable cases, complete sequence and glycan position information is obtained from a single-scan spectrum. The "mild" character of ECD supports the previously proposed non-ergodic (cleavage prior to energy randomization) mechanism, and the low internal energy increment of fragments.
Mirgorodskaya
E
E
Department of Molecular Biology, University of Southern Denmark/Odense University, Denmark.
Roepstorff
P
P
Zubarev
R A
RA
D000595:Amino Acid Sequence; D005583:Fourier Analysis; D006031:Glycosylation; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D013057:Spectrum Analysis
D010455:Peptides