Direct structural assignment of neutral and sialylated N‐glycans of glycopeptides using collision‐induced dissociation MS n spectral matching

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., GlcNAc β 1‐4GlcNAc, 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. Copyright © 2006 John Wiley & Sons, Ltd.