title: Fragmentation characteristics of deprotonated N-linked glycopeptides: influences of amino acid composition and sequence.
abstract: Glycopeptide structural analysis using tandem mass spectrometry is becoming a common approach for elucidating site-specific N-glycosylation. The analysis is generally performed in positive-ion mode. Therefore, fragmentation of protonated glycopeptides has been extensively investigated; however, few studies are available on deprotonated glycopeptides, despite the usefulness of negative-ion mode analysis in detecting glycopeptide signals. Here, large sets of glycopeptides derived from well-characterized glycoproteins were investigated to understand the fragmentation behavior of deprotonated N-linked glycopeptides under low-energy collision-induced dissociation (CID) conditions. The fragment ion species were found to be significantly variable depending on their amino acid sequence and could be classified into three types: (i) glycan fragment ions, (ii) glycan-lost fragment ions and their secondary cleavage products, and (iii) fragment ions with intact glycan moiety. The CID spectra of glycopeptides having a short peptide sequence were dominated by type (i) glycan fragments (e.g., (2,4)AR, (2,4)AR-1, D, and E ions). These fragments define detailed structural features of the glycan moiety such as branching. For glycopeptides with medium or long peptide sequences, the major fragments were type (ii) ions (e.g., [peptide + (0,2)X0-H](-) and [peptide-NH3-H](-)). The appearance of type (iii) ions strongly depended on the peptide sequence, and especially on the presence of Asp, Asn, and Glu. When a glycosylated Asn is located on the C-terminus, an interesting fragment having an Asn residue with intact glycan moiety, [glycan + Asn-36](-), was abundantly formed. Observed fragments are reasonably explained by a combination of existing fragmentation rules suggested for N-glycans and peptides.
authors.affiliation: Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan, nishikaz@shimadzu.co.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nishikaze
authors.forename: Shin-ichirou
authors.initials: S
authors.lastname: Kawabata
authors.forename: Koichi
authors.initials: K
authors.lastname: Tanaka
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D006020:Glycopeptides / Q000737:chemistry*; D007477:Ions / Q000737:chemistry; D013058:Mass Spectrometry; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D006020:Glycopeptides; D007477:Ions; D010446:Peptide Fragments; D011134:Polysaccharides