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

A method has been developed which allows the analysis of glycoproteins sepa rated by SDS-PAGE. The procedure, though applicable to N-glycosylated glyco proteins of any origin, is particularly devised for glycoproteins potential ly containing fucose in alpha 1,3-linkage to the reducing GlcNAc as may be found in plants and invertebrates, e.g., insects and parasitic helminths. S tarting with an established procedure for mass spectrometric peptide mappin g, 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 meth od was tested on proteins with N-glycans of known structure, i.e., as horse radish peroxidase, zucchini ascorbate oxidase, soybean agglutinin, honeybee venom hyaluronidase, bovine ribonuclease B, and bovine fetuin. An electrop horetic band corresponding to 4 Erg of glycoprotein was generally sufficien t to allow detection of the major N-glycan species. As an additional benefi t, a peptide mass map is generated which serves to identify the analyzed pr otein. The method was applied to glycoprotein allergens whose glycan struct ures were unknown. Ara h 1 and Ole e 1, major allergens from peanut and oli ve pollen, respectively, contained mainly xylosylated N-glycans with the co mposition Man(3(-4))XylGlcNAc(2) in the case of Ara h 1 and GlcNAc(1-2)-Man (3)XylGlcNAc(2) in the case of Ole e 1 where also some GlcNAc(0-2)Man(3)Xyl FucGlcNAc(2) was found. (C) 2000 Academic Press.