Localization of O-Glycosylation Sites in Peptides by Electron Capture Dissociation in a Fourier Transform Mass Spectrometer

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Abstract
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 nonergodic (cleavage prior to energy randomization) mechanism, and the low internal energy increment of fragments.