DETECTION OF POLYPEPTIDE ALTERATIONS DURING HEPATOCARCINOGENESIS

肝癌发生过程中多肽变化的检测

• 批准号: 5201569
• 负责人: P J WIRTH
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5201569
• 关键词: SDS polyacrylamide gel electrophoresis; carcinogenesis; clone cells; computer assisted sequence analysis; human fetus tissue; human tissue; liver cells; liver neoplasms; neoplastic transformation; nucleoproteins; peptide structure; protein sequence; tissue /cell culture

Simplified methodology has been developed for the direct N-terminal amino acid microsequencing of human liver and hepatoma-derived polypeptides using micropreparative immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis (IPG 2D-PAGE). Utilization of IPG gel strips in the first dimension permitted protein loading concentrations of 0.5-2.0 mg with negligible diminution of polypeptide resolution. Following 2D separation and electrotransfer to PVDF membranes, nearly 100 well resolved Ponceau S stained polypeptides were readily visualized on respective blotted membranes, from which 32 adult liver S-9 and 72 HepG2 nuclear cytosolic polypeptides were subjected to N-terminal microsequencing. Twenty normal adult liver and 54 HepG2 polypeptides yielded N-terminal sequence information, of which 17 and 19 polypeptides, respectively, exhibited high sequence homology to previously identified proteins. The initial yields of the proteins sequenced ranged from 2-14 pmols and yielded sequences of 14-26 amino acid residues. Many of the adult liver and HepG2 proteins contained inferred leader sequences since the first sequenced residue was several (20-30) residues from the methionine initiation site predicted by the cDNA. Comparison of approximately 1000 silver stained whole cell lysate and purified nuclear proteins between normal adult liver, two nontransformed cell lines [Chang (adult) and WRL-68 (embryonic)], and four human hepatoma- derived cell lines (HepG2, Huh-7, FOCUS, and SK-Hep) revealed significant qualitative and quantitative differences in polypeptide expression. Chang and WRL-68 liver cells, whose 2D polypeptide patterns were almost completely superimposable, most resembled normal liver, while marked differences in polypeptide expression were observed between normal liver and each of the hepatoma-derived cell lines (HepG2, FOCUS, Huh-7 and SK-Hep). Preparative IPG 2D-PAGE in combination with protein microsequencing provides a convenient one-step procedure to rapidly obtain partial amino acid sequence information for nearly 100 individual polypeptides directly from a single 2D-PAGE gel. Comparison of partial amino acid sequences with existing protein and nucleic acid sequence databases provide a rapid and convenient method of protein identification in the absence of specific antibody preparations as well as to suggest possible biological function(s) for as yet unidentified proteins.

简化的方法已被开发为直接的N-末端氨基 人肝和肝癌衍生多肽的酸性微测序 使用微制备固定化pH梯度二维 聚丙烯酰胺凝胶电泳（IPG 2D-PAGE）。 IPG的使用 在第一维中的凝胶条允许蛋白质加载 浓度为0.5-2.0 mg，多肽减少可忽略不计 分辨率 在2D分离和电转移到PVDF之后 膜，近100个良好解决丽春红S染色的多肽， 在各自的印迹膜上很容易观察到， 对肝S-9和72个HepG 2核胞质多肽进行处理， N-末端微测序。 20例正常成人肝和54例HepG 2 多肽产生了N-末端序列信息，其中17和19 多肽，分别表现出高度的序列同源性， 以前发现的蛋白质。 蛋白质的初始产量 测序范围为2-14 pmol，得到14-26个氨基酸的序列， 酸残留物。 许多成人肝脏和HepG 2蛋白含有 由于第一个测序的残基是几个 （20-30）个氨基酸残基， cDNA。 约1000份银染色全细胞裂解物的比较 和纯化的核蛋白之间的正常成人肝脏，两个 非转化细胞系[Chang（成体）和WRL-68（胚胎）]，以及 四种人肝癌细胞系（HepG 2、Huh-7、FOCUS和SK-Hep） 揭示了显着的定性和定量差异， 多肽表达。 Chang和WRL-68肝细胞，其二维 多肽模式几乎完全重叠，大多数 与正常肝脏相似，而多肽 在正常肝脏和每个 肝癌衍生的细胞系（HepG 2、FOCUS、Huh-7和SK-Hep）。 电泳IPG 2D-PAGE结合蛋白质微测序 提供了一种简便的一步法， 近100种多肽的氨基酸序列信息， 从单一的2D-PAGE凝胶。部分氨基酸序列比较 利用现有的蛋白质和核酸序列数据库， 和方便的蛋白质鉴定方法， 特异性抗体制剂以及提示可能的生物学 功能（s）尚未确定的蛋白质。