Electron Capture Dissociation of O-Glycosylated Peptides: Radical Site-Induced Fragmentation of Glycosidic Bonds

Abstract

Glycosylation of proteins represents one of the most important post-translational modifications. The structural characterisation of glycoproteins--especially with respect to the determination of the glycosylation site--by direct mass spectrometric methods still remains an elusive goal. We have applied the low energy dissociation method electron capture dissociation (ECD) in a 9.4 T Fourier transform ion cyclotron resonance mass spectrometer to the structural elucidation of mucin-derived peptides glycosylated with glycans of different core types. Capture of an electron by multiply protonated precursor ions [M + nH](n+) resulted in the formation of reduced odd electron radical cations [M + nH](n-1)+*. Subsequent cleavage of the N-Calpha bonds of the peptide chain, mostly without loss of the labile sugar moiety, represents a major fragmentation pathway allowing unambiguous assignment of the glycosylation site. In addition to peptide backbone cleavages, loss of acetyl radicals from the N-acetyl group of the HexNAc glycans is observed. Radical site induced elimination processes of the glycan moieties initiated by hydrogen transfer, from the glycan to the peptide backbone and vice versa give rise to signals in the ECD spectra. The different sugar core types exhibit different fragmentation patterns driven by the stability of the resulting fragments allowing the discrimination of isomeric glycans.

Human Urinary Glycoproteomics; Attachment Site Specific Analysis of N- and O-Linked Glycosylations by CID and ECD

“…2I). A plausible explanation for these secondary fragments has been attributed to the loss of an acetyl radical (C 2 H 3 O • ) from the N-acetyl moiety of HexNAc containing glycopeptides (53). Also, elimination of HexHexNAc from precursor ions was occasionally observed in ECD-MS 2 (m/z 1010.49 in Fig.…”

Electron detachment dissociation of glycosaminoglycan tetrasaccharides

“…The resulting radical is stabilized by delocalization with the oxygen that forms the glycosidic bond to C4. H-atom transfer from carbon atoms in saccharide rings has been observed in collision induced electron detachment mass spectra°of°nucleotides° [28],°in°reactions°between°phenyl radical°cations°and°ribose° [41],°and°in°the°ECD°mass spectra°of°glycopeptides° [42].°The°radical°site°at°C4°can promote 3,5 A 3 and 0,2 A 3 fragmentation as shown in Scheme 4. The abundance of cross ring cleavage products from fragmentation in the second sugar residue from the reducing end in the EDD spectrum, and their absence in the CAD and IRMPD spectra, suggests radical-induced fragmentation of many of the bonds in this residue, which can be rationalized by mobility of the radical site via hydrogen rearrangement.…”

The combination of electron capture dissociation and fixed charge derivatization increases sequence coverage for O-glycosylated and O-phosphorylated peptides

“…This fragmentation has been discussed°in°details°by°Mormann°et°al.° [27]°who°pro-posed a mechanism based on the attack of a hot hydrogen atom at the nitrogen of the N-acetyl group of one glycan leading to an N-centered hypervalent radical, which could subsequently fragment by loss of an acetyl radical (43.0178 Da). Nevertheless, it should be noted that the attack of the hydrogen atom should be more likely at the carbonyl oxygen which has a higher hydrogen affinity than the amide nitrogen and therefore this mechanism seems unlikely.…”