Bauw G, Rasmussen H H, van den Bulcke M, van Damme J, Puype M, Gesser B, Celis J E, Vandekerckhove J
Laboratorium voor Genetica, Rijksuniversiteit Gent, Belgium.
Electrophoresis. 1990 Jul;11(7):528-36. doi: 10.1002/elps.1150110703.
We have used two-dimensional gel electrophoresis as a general "preparative" method to purify proteins for microsequencing analysis. In the first experiments, proteins derived from a total extract of Nicotiana tabacum leaf tissue were directly blotted from the gel onto poly(4-vinyl-N-methylpyridinium iodide)-coated glass fiber sheets. The major spots were excised and subjected to NH2-terminal sequence analysis, which made it possible to identify five of the eight selected proteins, while two more were recognized by generated internal sequences. In a second set of experiments, proteins of human origin were separated on multiple two-dimensional gels and the Coomassie Brilliant Blue-stained spots were excised from the gels. The combined spots were re-eluted and concentrated in a new gel and blotted on Immobilon. They were fragmented by in situ proteolysis and the generated peptides were separated by reverse phase-high performance liquid chromatography and sequenced. At the average, the internal sequences that were obtained covered 35 residues per protein and allowed unambiguous identification of 13 of the 23 proteins analyzed so far. The sequence information obtained of the unidentified proteins is sufficient for further cloning. These results demonstrate that systematic sequence analysis of the major proteins seen in two-dimensional gels is within the reach of current technologies. This offers a unique opportunity to link information contained in protein databases with known or forthcoming DNA sequence data.
我们使用二维凝胶电泳作为一种通用的“制备性”方法来纯化蛋白质，以进行微量测序分析。在最初的实验中，将来自烟草叶片组织总提取物的蛋白质直接从凝胶印迹到涂有聚（4-乙烯基-N-甲基碘化吡啶）的玻璃纤维片上。切除主要斑点并进行氨基末端序列分析，这使得能够鉴定出所选的八种蛋白质中的五种，另外两种则通过产生的内部序列得以识别。在第二组实验中，将人源蛋白质在多个二维凝胶上进行分离，然后从凝胶上切下考马斯亮蓝染色的斑点。将合并的斑点重新洗脱并在新凝胶中浓缩，然后印迹到Immobilon上。通过原位蛋白酶解将它们片段化，产生的肽通过反相高效液相色谱分离并测序。平均而言，获得的内部序列每个蛋白质覆盖35个残基，并且能够明确鉴定出到目前为止分析的23种蛋白质中的13种。从未鉴定蛋白质获得的序列信息足以进行进一步的克隆。这些结果表明，对二维凝胶中可见的主要蛋白质进行系统的序列分析在当前技术范围内。这提供了一个独特的机会，将蛋白质数据库中包含的信息与已知或即将出现的DNA序列数据联系起来。
