Separation and characterization of nitrated variants of the major birch pollen allergen by CZE ‐ ESI ‐μ TOF MS

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 ( B et v 1a). In‐house nitration of recombinant B et 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 B et 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 p I 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 B et v 1a variants.