N-glycan analysis by matrix-assisted laser desorption/ionization mass spectrometry of electrophoretically separated nonmammalian proteins: Application to peanut allergen Ara h 1 and olive pollen allergen Ole e 1

Daniel Kolarich, Friedrich Altmann*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

147 Citations (Scopus)

Abstract

A method has been developed which allows the analysis of glycoproteins separated by SDS-PAGE. The procedure, though applicable to N-glycosylated glycoproteins of any origin, is particularly devised for glycoproteins potentially containing fucose in α1,3-linkage to the reducing GlcNAc as may be found in plants and invertebrates, e.g., insects and parasitic helminths. Starting with an established procedure for mass spectrometric peptide mapping, the analysis of N-glycans by matrix-assisted laser desorption/ionization mass spectrometry involved the use of peptide:N-glycosidase A, a triphasic microcolumn for sample cleanup, and a new matrix mixture consisting of 2,5-dihyhydroxybenzoic acid, 1-hydroxyisoquinoline, and arabinosazone. The method was tested on proteins with N-glycans of known structure, i.e., as horseradish peroxidase, zucchini ascorbate oxidase, soybean agglutinin, honeybee venom hyaluronidase, bovine ribonuclease B, and bovine fetuin. An electrophoretic band corresponding to 4 μg of glycoprotein was generally sufficient to allow detection of the major N-glycan species. As an additional benefit, a peptide mass map is generated which serves to identify the analyzed protein. The method was applied to glycoprotein allergens whose glycan structures were unknown. Ara h 1 and Ole e 1, major allergens from peanut and olive pollen, respectively, contained mainly xylosylated N-glycans with the composition Man(3(-4))XylGlcNAc2 in the case of Ara h 1 and GlcNAc1-2-Man3XylGlcNAc2 in the case of Ole e 1 where also some GlcNAc0-2Man3XylFucGlcNAc2 was found. (C) 2000 Academic Press.

Original languageEnglish
Pages (from-to)64-75
Number of pages12
JournalAnalytical Biochemistry
Volume285
Issue number1
DOIs
Publication statusPublished - 1 Oct 2000

Fingerprint

Dive into the research topics of 'N-glycan analysis by matrix-assisted laser desorption/ionization mass spectrometry of electrophoretically separated nonmammalian proteins: Application to peanut allergen Ara h 1 and olive pollen allergen Ole e 1'. Together they form a unique fingerprint.

Cite this