Division of Chemistry and Bioanalytics, Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
Electrophoresis. 2013 Sep;34(18):2695-704. doi: 10.1002/elps.201300151. Epub 2013 Aug 21.
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.
已经优化了 CZE-ESI-TOF MS 方法，用于分离和鉴定来自欧洲桦树花粉过敏原 1a 异构体（Bet v 1a）的主要异源硝化变体。通过过氧亚硝酸盐对重组 Bet v 1a 进行了内源性硝化。作为 BGE，pH 值为 7.50 的 10 mmol/L 碳酸氢铵提供了最佳的分辨率。分别为 0.4 巴和 6 μL/min 的雾化器气体压力和鞘液流速维持了 CZE 的选择性，并构成了稳定的电喷雾条件。在超纯水中含有 75% v/v 甲醇和 0.1% v/v 甲酸的鞘液组成可获得最高的信号强度。或者，甲醇可以被 50% v/v 的异丙醇取代。通过详细的 CZE-ESI-TOF MS 方法比较了含有不同量硝化试剂的反应混合物中两种改良的过敏原产物。多达十二种不同的 Bet v 1a 变体，硝化程度为一至六倍，可以区分开来。几个等效硝化等级的过敏原部分得到了分离。它们不同的迁移时间表明存在特定部位的硝化，同时伴随着 pI 和可能还有流体力学半径的差异。该方法允许对内部硝化的过敏原样品进行表征，这些样品旨在测试硝化的 Bet v 1a 变体假定的增强致敏性。
