美洲大蠊主要变应原蛋白的质谱鉴定与分析

为了建立美洲大蠊Periplaneta americana变应原蛋白的质谱鉴定方法，我们将美洲大蠊粗浸液通过DEAE-52离子交换层析、Sephacryl S-200凝胶过滤层析等分离步骤得到纯化的74 kD蛋白，对纯化前后的该74 kD蛋白分别进行SDS-PAGE及凝胶内胰酶酶切，再经液相色谱-电喷雾-串联质谱（HPLC-ESI-MS/MS）在线联机分析，所得质谱数据进入网站（http://www.matrixscience.com）进行Mascot检索比对。通过对两者质谱鉴定结果的比较来评估美洲大蠊天然主要变应原蛋白的纯化效果。结果表明，纯化蛋白经HPLC-ESI-MS/MS鉴定是美洲大蠊主要变应原蛋白；离子交换层析等纯化步骤可以去除同一分子量的杂蛋白(如卵黄原蛋白)，从而获得较好的鉴定结果。我们首次成功地运用质谱建立起变应原蛋白的新鉴定方法。

Identification of the major allergen in Periplaneta americana by HPLC-ESI-MS/MS

A new method to identify allergen of Periplaneta americana with HPLC ESI MS/MS was established. The protein with molecular weight of 74 kD from P. americana was purified by sequential chromatography of two steps including ion exchange chromatography and gel filtration chromatography. After in gel digestion of allergen,the tryptic peptide mapping of allergen was obtained by HPLC ESI MS/MS. The data were then searched against NCBInr database using MS/MS Ions search (http://www.matrixscience.com). We compared the results of identification of 74 kD protein from the crude extract of P. americana and from the purified protein to evaluate the effect of purification. Our data showed that the purified protein with molecular weight of 74 kD is the major allergen of P. americana. The steps of chromatography are effective to remove the other protein with the same size as the purified protein. To our knowledge, this is the first report on the identification of the major allergen from P. americana by HPLC ESI MS/MS. The research indicated that the MS is simple and universal for the quality control of naturally purified allergen.