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Fragmentation Characteristics of Deprotonated N-linked Glycopeptides: Influences of Amino Acid Composition and Sequence

  • Research Article
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Journal of The American Society for Mass Spectrometry

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., 2,4AR, 2,4AR-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 + 0,2X0–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.

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Acknowledgments

The authors are grateful to Dr. Sekiya for his valuable comments and suggestions. This research is supported by the Japan Society for the Promotion of Science (JSPS) through its Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program).

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  1. Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan

    Takashi Nishikaze, Shin-ichirou Kawabata & Koichi Tanaka

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  1. Takashi Nishikaze
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  2. Shin-ichirou Kawabata
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Correspondence to Takashi Nishikaze.

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Nishikaze, T., Kawabata, Si. & Tanaka, K. Fragmentation Characteristics of Deprotonated N-linked Glycopeptides: Influences of Amino Acid Composition and Sequence. J. Am. Soc. Mass Spectrom. 25, 988–998 (2014). https://doi.org/10.1007/s13361-014-0854-7

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