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

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 stuctural 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–Cα 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.