Ｏグリコプロテオームと電子移動解離ＭＳ

抄録

Structural analysis of O-glycopeptides is much more challenging than N-glycosylation due to a number of difficulties; There are more than fifteen glycosyl enzymes (GalNTs) in human for initiating O-glycosylation of proteins, and no consensus sequence for O-glycosylation has been described. The assignment of attachment sites of the glycan is further complicated by the fact that there may be many sites of O-glycan attachment adjacent to each other and that these are frequently located in a region of peptide sequence in which many serine and threonine residues are found together, all of which may or may not be glycosylated. In addition, proline residues are often present in the sequence and hamper the sequencing with MS/MS, due to the high efficiency of cleavage at the amide bond on the N-terminal of proline residue in collision-induced dissociation (CID). As a result, quite a small number of attachment sites have been experimentally determined and recorded in the protein database to date.
In our O-glycoproteome project, various chemical methods such as b-elimination followed by Michael addition of different kinds of substituents and partial deglycosylation with trifulromethanesulfonic acid were examined, but they were inadequate to solve the problems when combined with CID. Electron capture dissociation and a similar technique electron transfer dissociation (ETD) produce significantly different types of fragment ions than other fragmentation modes including CID, which introduces internal vibrational energy. In ECD and ETD, labile post-translational modifications such as phosphorylation, glycosylation and others remain attached to the backbone during ECD or ETD MS/MS experiments allowing determination of the site and identity of post-translational modifications. Indeed, we have determined a number of new O-glycosylation sites of plasma proteins with the aid of ETD.