基于8-氨基1,3,6-萘三磺酸二钠盐的糖基化蛋白质标记方法

Research on 8-aminonaphthalene 1,3,6-trisulfonic acid disodium salt based fluorescent tag for selective glycoprotein labeling

发展了基于荧光试剂8-氨基1,3,6-萘三磺酸二钠盐(ANTS)的糖基化蛋白质选择性标记和荧光检测的新方法.实验中以标准蛋白质细胞色素c和核糖核酸酶b为样品,优化了pH和反应时间.在优化的条件下,核糖核酸酶b的检测灵敏度可提高22倍,而非特异性的标记仅为1%,显示了这种衍生方法极好的选择性.进一步将该方法应用于卵清蛋白的衍生,通过结合二氧化钛(TiO2)亲和富集,共鉴定到14个蛋白质,其中超过85%的蛋白质具有一致的N-X-T/S(X为除脯氨酸以外的其他氨基酸)序列,为潜在的糖基化蛋白,进一步验证了该方法的高选择性.

The selective glycoprotein labeling method based on fluorescent tag 8-aminonaphthalene-1,3,6-trisulfonic acid disodium （ANTS） was developed in the present work.With proteins cytochrome c and ribonuclease b as the samples,the pH value and reaction time were optimized.The fluorescent detection sensitivity of glycoprotein ribonuclease b could be increased about 22-fold via ANTS labeling while the nonspecific labeling only 1％,indicating the good selectivity of the developed method.The method was further used for derivatization of egg white proteins.By combining with titanium dioxide （TiO2） for glycoproteins enrichment,totally 14 proteins were identified,of which 85％ proteins were recognized with the sequence N-X-T/S （X except Pro）,as the potential glycoproteins,which further indicated the high selectivity of the developed method.All these results suggested the potential merits of the developed method for high specific glycoprotein recognition.