Structural analysis of N-linked oligosaccharides by a combination of glycopeptidase, exoglycosidases, and high-performance liquid chromatography

https://doi.org/10.1016/0003-2697(87)90253-3Get rights and content

Abstract

A simple, sensitive, and rapid method for the analysis of structures of N-linked carbohydrates is reported. The method involves four steps: (i) preparation of carbohydrate chains from glycopeptides by N-oligosaccharide glycopeptidase digestion; (ii) derivatization of the reducing ends of carbohydrate chains with a fluorescent reagent, 2-aminopyridine, by using sodium cyanoborohydride; (iii) separation of oligosaccharide derivatives by reverse-phase high-performance liquid chromatography; and (iv) structural analysis of oligosaccharides by sequential exoglycosidase digestion. The elution positions of 50 standard oligosaccharide derivatives were determined by HPLC. The structure of an unknown oligosaccharide can be characterized by comparison of its elution position with those of the standard compounds. The method was applied to elucidate the structures of oligosaccharides in the myeloma IgG protein, Yot.

References (20)

  • N. Takahashi

    Biochem. Biophys. Res. Commun

    (1977)
  • N. Takahashi et al.

    Biochim. Biophys. Acta

    (1981)
  • H. Ishihara et al.

    Biochem. Biophys. Res. Commun

    (1983)
  • A.L. Tarentino et al.

    J. Biol. Chem

    (1982)
  • H. Yoshima et al.

    J. Biol. Chem

    (1981)
  • T. Tai et al.

    J. Biol. Chem

    (1975)
  • S. Takasaki et al.
  • H. Nishibe et al.

    Biochim. Biophys. Acta

    (1981)
  • T. Hotta et al.

    J. Appl. Biochem

    (1985)
  • N. Takahashi et al.

    Biochemistry

    (1987)
There are more references available in the full text version of this article.

Cited by (113)

  • Reversed-phase and hydrophobic interaction chromatography of carbohydrates and glycoconjugates

    2021, Carbohydrate Analysis by Modern Liquid Phase Separation Techniques
  • Analysis of 2-aminopyridine labeled glycans by dual-mode online solid phase extraction for hydrophilic interaction and reversed-phase liquid chromatography

    2020, Journal of Chromatography A
    Citation Excerpt :

    Glycan separation on HILIC phases is the most popular, and the separation was primarily governed by the hydrophilic nature of glycans [15]. Separation based on differences in linkage and the monosaccharide components of glycans was obtained on a reversed phase column [16], and the retention data of a few hundred glycoprotein glycans have been compiled into a website database [17]. The choice of labeling reagent is important in reversed-phase separation.

  • The underestimated N-glycomes of lepidopteran species

    2017, Biochimica et Biophysica Acta - General Subjects
    Citation Excerpt :

    Considering that insect glycolipids with a GlcAβ1,3Galβ1,3GalNAcα1,4GalNAcβ1,4GlcNAcβ-R motif are known [39], the 10 g.u. product was then treated with chicken liver α-N-acetylgalactosaminidase and loss of one HexNAc residue was observed together with a shift to 9.5 g.u. (m/z 1541) and the replacement of the m/z 610 B fragment by one of m/z 407 (HexdiNAc; Fig. 9E). As the first intermediate product co-eluted with the m/z 2082 glycan found in the original glycome, these data also can be taken as fixing its structure; as the final product of m/z 1541 of these digestions is known to have a ‘lower’ arm due to its elution time [27], the complex antenna is concluded to be on the α1,3-mannose, while the later-eluting m/z 2247 isomer (Fig. 8N) is carrying this antenna probably on the α1,6-mannose as ‘upper’ arm modifications tend to result in increased retention on RP-type columns [40]. A similar approach was used to resolve the L. dispar m/z 2044 glycan eluting at 7.6 g.u. (HexA1Hex4HexNAc4Fuc1PC1; see Figs. 8M and 9G for negative and positive mode MS/MS).

  • Multistep fractionation and mass spectrometry reveal zwitterionic and anionic modifications of the N- and O-glycans of a marine snail

    2016, Molecular and Cellular Proteomics
    Citation Excerpt :

    In the case of V. rubella, the glycans carrying sulfate or glucuronylated fucose were primarily in the “anionic” pool, whereas the “hydrophobic” pool is very much biased toward structures with core Fucα1,2Galβ1,4Fucα1,6 motifs. The subsequent use of RP-HPLC facilitates the separation of isomeric and isobaric structures, a strategy established for some 30 years to resolve complex, paucimannosidic, and oligomannosidic glycans from mammalian and other sources (29). Another aspect, which we consider important, is the optimized mix of methods on individual RP-HPLC fractions.

View all citing articles on Scopus
View full text