title: Direct structural assignment of neutral and sialylated N-glycans of glycopeptides using collision-induced dissociation MSn spectral matching.
abstract: Mass spectrometric analyses of various N-glycans binding to proteins and peptides are highly desirable for elucidating their biological roles. An approach based on collision-induced dissociation (CID) MS(n) spectra acquired by electrospray ionization linear ion trap time-of-flight mass spectrometry (ESI-LIT-TOFMS) in the positive- and negative-ion modes has been proposed as a direct method of assigning N-glycans without releasing them from N-glycopeptides. In the positive-ion mode of this approach, the MS(2) spectrum of N-glycopeptide was acquired so that a glycoside-bond cleavage occurs in the chitobiose residue (i.e., GlcNAcbeta1-4GlcNAc, GlcNAc: N-acetylglucosamine) attached to asparagine (N), and two charges on the [M+H+Na](2+) precursor ion are shared with both of the resulting fragments. These fragments are sodiated B(n)-type fragment ions of oligosaccharide (N-glycan) and a protonated peptide ion retaining one GlcNAc residue on the asparagine (N) residue. The structure of N-glycan was assigned by comparing MS(3) spectra derived from both the sodiated B(n)-type fragment ions of N-glycopeptide and the PA (2-aminopyridine) N-glycan standard (i.e., MS(n) spectral matching). In a similar manner, the structural assignment of sialylated N-glycan was performed by employing the negative-ion CID MS(n) spectra of deprotonated B(n)-type fragment ions of N-glycopeptide and the PA N-glycan standard. The efficacy of this approach was tested with chicken egg yolk glycopeptides with a neutral and a sialylated N-glycan, and human serum IgG glycopeptides with neutral N-glycan isomers. These results suggest that the approach based on MS(n) spectral matching is useful for the direct and simple structural assignment of neutral and sialylated N-glycans of glycopeptides.
authors.affiliation: Graduate School of Advanced Life Science, Hokkaido University, Sapporo 001-0021, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Ito
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Takegawa
authors.forename: Kisaburo
authors.initials: K
authors.lastname: Deguchi
authors.forename: Shinji
authors.initials: S
authors.lastname: Nagai
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Nakagawa
authors.forename: Yasuro
authors.initials: Y
authors.lastname: Shinohara
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Nishimura
mesh_terms: D000465:Algorithms; D000818:Animals; D002645:Chickens; D006020:Glycopeptides / Q000032:analysis* / Q000097:blood / Q000737:chemistry*; D006801:Humans; D011134:Polysaccharides / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D006020:Glycopeptides; D011134:Polysaccharides