质谱法分析大肠癌组织中踝蛋白的磷酸化修饰

踝蛋白的磷酸化修饰,特别是肿瘤等病理条件下的踝蛋白磷酸化状态,与肿瘤的发病、转移机理密切相关。本研究采用盐析、离子交换层析和电泳分离并纯化了人大肠癌组织中的踝蛋白(Talin),经胰酶水解获得其肽段混合物,进一步分别利用固定化Fe3+亲和层析和TiO2亲和层析在酸性条件下对其中磷酸化修饰肽段进行吸附,并以1%氨水进行洗脱。在Michrom Magic C18色谱柱上,以A:99%水+1%乙腈+0.1%甲酸和B:99%乙腈+1%水+0.1%甲酸两种流动相进行梯度洗脱分离,采用ESI质谱进行依赖数据的二级子离子扫描。结果显示,固定化Fe3+富集到8个磷酸化肽段而TiO2富集到9个磷酸化肽段。本研究提供了一种快速、准确地鉴定从人大肠癌组织中分离表征踝蛋白的方法。

Mass Spectrometric Analysis of Phosphorylation Modification in Talin from Human Colorectal Carcinoma Tissues

The phosphorylation modification of talin,especially the phosphorylation state of talin in pathological environment such as carcinoma,is closely relevant to carcinogenesis and metastasis process.In this study,talin protein was isolated from human colorectal carcinoma tissues by salt fractionation and ion exchange chromatography,followed by further purification by electrophoresis.The purified talin was subject to tryptic digestion.Either immobilized Fe3+ affinity chromatography or TiO2 affinity chromatography was used to absorb the phosphorylated peptides under acidic condition,which were then eluted with 1% ammonium hydroxide.The separation was performed on a Michrom Magic C18 column with gradient elution using two mobile phase solutions(A:99% water+1% ACN+0.1% formic acid;B:99% ACN+1% water+0.1% formic acid).ESI mass spectrometer was used to detect the product ions of the eluted peaks under data-dependent acquisition mode.The results indicated that 8 phosphorylated peptides were captured by the immobilized Fe3+ affinity chromatography,whereas 9 phosphorylated peptides were captured by TiO2 affinity chromatography.The present study provides a rapid,accurate method for characterizing talin protein isolated from human colorectal carcinoma tissues.