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Protein-Specific Analysis of Invertebrate Glycoproteins

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Functional Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1871))

Abstract

N-Glycans are posttranslational modifications of proteins attached to the amide side chains of asparagine residues, with possible heterogeneity due to different structures being possible at the same glycosylation site. In contrast to the mammalian systems, invertebrate N-glycosylation presents a challenge in analysis as there exist unfamiliar epitopes and a high degree of structural and isomeric variation between different species. A simple analytical approach to analyze N-glycans on specific glycoproteins is presented, which involves a combination of tryptic peptide mass spectrometry and “off-line” RP-HPLC MALDI-TOF MS/MS complemented by blotting to recognize specific epitopes. An additional N-glycan enrichment and labeling step can facilitate the analysis of single structures and even provide isomeric separation of N-glycans from specific proteins.

Full data on the N-glycans of honeybee royal jelly will appear in a forthcoming paper: Hykollari et al. 2018. Isomeric separation and recognition of anionic and zwitterionic N-glycans from royal jelly glycoproteins.

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Abbreviations

DTT:

Dithiothreitol

HRP:

Horseradish peroxidase

MALDI-TOF:

MS matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry

NPGC:

Nonporous graphitized carbon

PC:

Phosphorylcholine

PE:

Phosphoethanolamine

RP-HPLC:

Reversed phase high pressure liquid chromatography

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

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Acknowledgments

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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Authors and Affiliations

  1. Department für Chemie, Universität für Bodenkultur, Vienna, Austria

    Alba Hykollari, Daniel Malzl, Iain B. H. Wilson & Katharina Paschinger

Authors
  1. Alba Hykollari
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  2. Daniel Malzl
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  3. Iain B. H. Wilson
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  4. Katharina Paschinger
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Corresponding author

Correspondence to Katharina Paschinger .

Editor information

Editors and Affiliations

  1. Array Bridge Inc., St. Louis, MO, USA

    Xing Wang

  2. Biotech Support Group LLC, Monmouth Junction, NJ, USA

    Matthew Kuruc

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Hykollari, A., Malzl, D., Wilson, I.B.H., Paschinger, K. (2019). Protein-Specific Analysis of Invertebrate Glycoproteins. In: Wang, X., Kuruc, M. (eds) Functional Proteomics. Methods in Molecular Biology, vol 1871. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8814-3_24

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  • DOI: https://doi.org/10.1007/978-1-4939-8814-3_24

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8813-6

  • Online ISBN: 978-1-4939-8814-3

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