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Analysis of Invertebrate and Protist N-Glycans

  • Alba Hykollari
  • Katharina Paschinger
  • Barbara Eckmair
  • Iain B. H. WilsonEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1503)

Abstract

N-glycans from invertebrates and protists have often unusual structures which present analytical challenges. Both core and antennal modifications can be quite different from the more familiar vertebrate glycan motifs; thereby, contrary to the concept that “simple” organisms have “simple” N-glycans, rather complex oligosaccharides structures, including zwitterionic and anionic ones, have been found in a range of species. Thus, to facilitate the optimized elucidation of the maximal possible range of structures, the analytical workflow for glycomics of these organisms should include sequential release and fractionation steps. Peptide:N-glycosidase F is sufficient to isolate N-glycans from fungi and some protists, but in most invertebrates core α1,3-fucose is present, so release of the glycans from glycopeptides with peptide:N-glycosidases A is required. Subsequent solid-phase extraction with graphitized carbon and reversed phase resins enables different classes of N-glycans to be separated prior to high-pressure liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Depending on the types and numbers of glycans present, either reversed- or normal-phase HPLC (or both in series) enable even single isomeric or isobaric structures to be separated prior to MALDI-TOF MS and MS/MS. The use of enzymatic or chemical treatments allows further insights to be gained, although some glycan modifications (especially methylation) are resistant. Using a battery of methods, sometimes up to 100 structures from a single organism can be assigned, a complexity which raises evolutionary questions regarding the function of these glycans.

Key words

Glycosylation Glycome Mass spectrometry Phosphorylated glycans Sulphated glycans 

Notes

Acknowledgment

This work was supported by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (FWF; grants P26662, P25058, and P23922 to A.H., K.P., and I.B.H.W.).

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Alba Hykollari
    • 1
  • Katharina Paschinger
    • 1
  • Barbara Eckmair
    • 1
  • Iain B. H. Wilson
    • 1
    Email author
  1. 1.Department für ChemieUniversität für BodenkulturViennaAustria

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