Bini L, Heid H, Liberatori S, Geier G, Pallini V, Zwilling R
Department of Molecular Biology, University of Siena, Italy.
Electrophoresis. 1997 Mar-Apr;18(3-4):557-62. doi: 10.1002/elps.1150180337.
Employing isoelectric focusing on immobilized pH gradients followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) we have obtained a map of C. elegans proteins, from a mixed culture containing all developmental stages, presenting over 2000 spots within the window of isoelectric points (pI) 3.5-9 and a molecular mass of 10-200 kDa. Edman microsequencing yielded successful results in 12 out of 24 analyzed spots. All but one of the N-terminal sequences retrieved C. elegans sequences in cosmid and/or expressed sequence tag clones. Structurally related protein sequences found in data banks included enzymes in energy metabolism (cytochrome oxydase, ATP synthase, enolase), a fatty acid-binding protein, a translationally controlled tumor protein, an unknown C. elegans protein, an acidic ribosomal protein, a titin-like protein, a G-protein beta chain, cyclophilin, and cathepsin D. Experimental determination of N-termini allowed us to define sites of signal cleavage providing further information on the physiological role of the newly found C. elegans proteins. This report demonstrates the possibility of two-dimensional gel electrophoresis and Edman microsequencing in the elucidation of C. elegans proteome.
采用固定化pH梯度等电聚焦，随后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE），我们从包含所有发育阶段的混合培养物中获得了秀丽隐杆线虫蛋白质图谱，在等电点（pI）3.5 - 9和分子量10 - 200 kDa的范围内呈现出2000多个斑点。在分析的24个斑点中，有12个通过埃德曼微量测序获得了成功结果。除了一个之外，所有检索到的N端序列在黏粒和/或表达序列标签克隆中均为秀丽隐杆线虫序列。数据库中发现的结构相关蛋白质序列包括能量代谢中的酶（细胞色素氧化酶、ATP合酶、烯醇化酶）、一种脂肪酸结合蛋白、一种翻译控制肿瘤蛋白、一种未知的秀丽隐杆线虫蛋白、一种酸性核糖体蛋白、一种肌联蛋白样蛋白、一种G蛋白β链、亲环蛋白和组织蛋白酶D。N端的实验测定使我们能够确定信号切割位点，从而为新发现的秀丽隐杆线虫蛋白质的生理作用提供进一步信息。本报告证明了二维凝胶电泳和埃德曼微量测序在阐明秀丽隐杆线虫蛋白质组方面的可能性。
