title: Separation and characterization of nitrated variants of the major birch pollen allergen by CZE-ESI-μTOF MS.
abstract: A CZE-ESI-TOF MS method has been optimized for the separation and identification of nitrated variants of the major birch pollen allergen from Betula verrucosa, isoform 1a (Bet v 1a). In-house nitration of recombinant Bet v 1a was done by peroxynitrite. As a BGE, 10 mmol/L ammonium bicarbonate with pH 7.50 provided best resolution. Nebulizer gas pressure and sheath liquid flow rate of 0.4 bar and 6 μL/min, respectively, maintained CZE selectivity and constituted stable electrospray conditions. A sheath liquid composition of 75% v/v methanol with 0.1% v/v formic acid in ultrapure water resulted in highest signal intensities. Alternatively, methanol could be replaced by 50% v/v isopropanol. Two modified allergen products derived from reaction mixtures that contained different amounts of the nitration reagent were compared by the elaborated CZE-ESI-TOF MS method. Up to twelve different Bet v 1a variants with one- to sixfold nitration could be distinguished. Several allergen fractions of equivalent nitration grade were resolved. Their different migration times indicate site-specific nitration with concomitant differences in pI and maybe also in hydrodynamic radius. The method allows for a characterization of in-house nitrated allergen samples that are intended for testing the postulated enhanced allergenicity of nitrated Bet v 1a variants.
authors.affiliation: Division of Chemistry and Bioanalytics, Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Sergey
authors.initials: S
authors.lastname: Gusenkov
authors.forename: Chloé
authors.initials: C
authors.lastname: Ackaert
authors.forename: Hanno
authors.initials: H
authors.lastname: Stutz
mesh_terms: D000485:Allergens / Q000737:chemistry*; D029662:Betula / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D009330:Nebulizers and Vaporizers; D009566:Nitrates / Q000032:analysis* / Q000737:chemistry; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry; D011058:Pollen / Q000737:chemistry*; D020033:Protein Isoforms / Q000032:analysis / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
chemical_list: D000485:Allergens; D009566:Nitrates; D010940:Plant Proteins; D020033:Protein Isoforms
keywords: Allergen; CZE; ESI-TOF MS; Nitration; Peroxynitrite