Structural studies on protein O-fucosylation by electron capture dissociation

Abstract

The low energy dissociation technique electron capture dissociation has been applied to a series of thrombospondin and properdin derived O-fucosylated glycopeptides. Followed by capture of an electron by multiply protonated precursor ions [M+ nH] n+ reduced odd electron radical cations [M+ nH] ( n −1) + were generated. The latter mainly fragment by cleavage of the NCα bonds of the peptide chain without loss of the labile sugar moiety allowing an unambiguous assignment of the glycosylation site. Apart from peptide backbone cleavages, side chain losses of aminocarbonylmethyl and aminocarbonylmethylthiyl radicals from carboxyamidomethylated cysteins are observed. The NCα bond cleavage is greatly reduced on both sides of alkylated Cys. However, fragment ions that are formed by secondary fragmentations of z-type radical cations containing N-terminal cystein give rise to even electron z SCH 2CONH 2 ions. The potential of the high mass accuracy for the identification of the protein modification topology has been fully explored.