DETECTION OF POLYPEPTIDE ALTERATIONS DURING HEPATOCARCINOGENESIS

双向聚丙烯酰胺凝胶电泳（2D-PAGE） RLE细胞多肽的数据库已经扩展到包括 关于1100个核质（胞质）和850个 微粒相关[35S]-蛋氨酸标记以及215 核质和269颗粒相关[32 P]-正磷酸标记 核多肽。 n-末端氨基酸 直接从对应的2D-PAGE凝胶进行多肽的微测序 到5 pmol的蛋白质常规地产生以下的可靠序列信息： 10 - 15个氨基酸残基。 银染全细胞比较 来自成人全肝组织的裂解物和纯化的核多肽 表明，高度的多肽模式相似性（80 - 90%）， 全细胞裂解物和纯化的成人全细胞核制备物 肝组织和来自非致瘤性Chang和WRL-68细胞系。 在正常肝和肝硬化患者之间观察到明显的多肽差异。 致瘤性HepG2和Huh-7细胞系。HepG2的2D-PAGE图谱 而Huh-7细胞几乎是完全不可能的 2D-PAGE结果 表明Chang和WRL-68细胞提供了有价值的体外系统 用于随后的详细生化研究，包括细胞周期 调节、生长因子调节、信号转导和凋亡， 在正常肝组织和肿瘤肝组织中。 计算机联机 Elsie 5分析系统与SWISS 2D-PAGE人肝蛋白 图谱显示> 80%的蛋白质匹配，包括以前的50种 表征的多肽。 为了补充瑞士2D-PAGE 人肝脏参考图谱，72个N-末端微序列信息 已获得HepG2核胞质多肽。十九 与已知蛋白质的氨基酸序列一致，35个氨基酸序列与已知蛋白质的氨基酸序列一致， 测序数据，但在PIR或SWISSPROT数据库中没有发现， 而18个是N-末端封闭的。 这些结果表明， 目前高度的微测序灵敏度和效率 可用，可以鉴定大百分比的多肽 直接从2D凝胶中提取。

The master 2-dimensional-polyacrylamide gel electrophoresis (2D-PAGE) database of RLE cellular polypeptides has been expanded to include detailed information concerning 1100 nucleoplasmic (cytosolic) and 850 particulate associated [35S]-methionine labeled as well as 215 nucleoplasmic and 269 particulate associated [32P]-orthophosphate labeled nuclear polypeptides, respectively. The N-terminal amino acid microsequencing of polypeptides directly from 2D-PAGE gels corresponding to 5 pmol of protein routinely yielded reliable sequence information of 10-15 amino acid residues. Comparison of silver stained, whole cell lysate and purified nuclear polypeptides from adult whole liver tissue indicated a high degree of polypeptide pattern similarity (80-90%) in whole cell lysate and purified nuclear preparations from adult whole liver tissue and from the non-tumorigenic Chang and WRL-68 cell lines. Marked polypeptide differences were observed between normal liver and the tumorigenic HepG2 and Huh-7 cell lines. The 2D-PAGE patterns of the HepG2 and Huh-7 cells were almost completely super-impossible. 2D-PAGE results suggest that the Chang and WRL-68 cells provide valuable in vitro systems for subsequent detailed biochemical studies including cell cycle regulation, growth factor modulation, signal transduction, and apoptosis, in normal versus neoplastic liver tissues. On-line computer linkage of the Elsie 5 analysis system with the SWISS 2D-PAGE human liver protein map revealed >80% matching of proteins, including 50 previously characterized polypeptides. In order to complement the SWISS 2D-PAGE human liver reference map, N-terminal microsequence information of 72 HepG2 nuclear cytosolic polypeptides has been obtained. Nineteen corresponded to known proteins, 35 yielded N-terminal amino acid sequencing data but were not found in the PIR or SWISSPROT databases, while 18 were N-terminally blocked. These results suggest that with the high degree of microsequencing sensitivity and efficiency currently available, it is possible to identify a large percentage of polypeptides directly from 2D-gels.