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 gen erated [M + nH]nt polypeptide cations produces rapid cleavage of the backbo ne NH-C, bond to form c and z ions (in the modified notation of Roepstorff and Fohlman). The potential of the Fourier transform mass spectrometry equi pped with ECD in structure analysis:of O-glycosylated peptides in the 3 kDa range has been investigated. Totally, 85% of the available interresidue bo nds were cleaved in five glycopeptides; more stable c ions accounted for 62 % of the observed fragmentation. The c series provided direct evidence on t he glycosylation sites:in every case studied, with no glycan (GalNAc and di mannose) losses observed from these species. Less stable z lions Supported the glycan site assignment, with minor glycan,: detachments. These losses, as well as the observed formation of even-electron z ions, are attributed t o radical-site-initiated reactions. In favorable cases, complete sequence a nd glycan position information is obtained from a single-scan spectrum. The "mild" character of ECD supports the previously proposed nonergodic (cleav age prior to energy randomization) mechanism, and the:low internal energy i ncrement of fragments.