磷酸化蛋白质组学鉴定 PTPLAD1调控的结肠癌细胞酪氨酸磷酸化蛋白质

摘要

目的：用磷酸化蛋白质组学的方法鉴定并分析结肠癌细胞中含蛋白酪氨酸磷酸酶样A结构域蛋白1（ PTPLAD1）调控的酪氨酸磷酸化蛋白。方法：运用siRNA技术沉默PTPLAD1的表达，于细胞培养条件下运用氨基酸稳定同位素标记技术（ stable lsotope labeling with amino acids in cell culture， SILAC）标记细胞，用酪氨酸磷酸化抗体免疫沉淀富集酪氨酸磷酸化蛋白，用LTQ-OrbitrapXL质谱鉴定因敲低PTPLAD1所出现的差异表达的酪氨酸磷酸化蛋白，进一步利用Ingenuity Pathway Analysis （ IPA）软件对这些差异蛋白进行生物信息学分析。结果：用real-time PCR筛选得到有效的siRNA干扰片段，Western blot验证其干扰效果及有效干扰时间。质谱鉴定PTPLAD1调控的差异酪氨酸磷酸化蛋白共20个，其中显著上调的8个，显著下调的10个，主要为转录因子及肿瘤标志物相关蛋白。 IPA软件的结果表明PTPLAD1调控的差异酪氨酸磷酸化蛋白的功能主要与器官发育分化、维持组织分化类型及细胞凋亡、增殖相关。结论：成功鉴定出PTPLAD1调控的差异酪氨酸磷酸化蛋白，可以为后续研究PTP-LAD1在结肠癌的发生发展中的作用及机理提供基础。%AIM:To identify and analyze tyrosine-phosphorylated proteins regulated by protein tyrosine phos-phatase-like A domain containing protein 1 ( PTPLAD1) in colon cancer cells by phosphoproteomics.METHODS: The expression of PTPLAD1 in colon cancer cell line HCT-116 was knocked down by small interfering RNAs, and the differenti-al expression of tyrosine-phosphorylated proteins in response to the konckdown of PTPLAD1 in HCT-116 cells was identified by stable isotope labeling with amino acid in cell culture ( SILAC) , coupled with the tyrosine phosphorylation antibody im-munoprecipitation and LC-MS/MS analysis.The Ingenuity Pathway Analysis ( IPA) software was employed for bioinformat-ics analysis on the differentially-expressed proteins.RESULTS:A total of 20 differentially-expressed tyrosine-phosphoryla-ted proteins were identified by MS, including 8 markedly up-regulated and 10 evidently down-regulated proteins.IPA soft-ware suggested that these proteins were mainly associated with the disease of cancer, tissue development and function, and cell death and survival.CONCLUSION:We successfully identified PTPLAD1-regulated differentially-expressed tyrosine-phosphorylated proteins in colon cancer cell line HCT-116.Our analysis suggests that PTPLAD1-regulated proteins in colon cancer are closely correlated with colon cancer.