Büttner K, Bernhardt J, Scharf C, Schmid R, Mäder U, Eymann C, Antelmann H, Völker A, Völker U, Hecker M
Institut für Mikrobiologie, Ernst-Moritz-Arndt-Universität Greifswald, Germany.
Electrophoresis. 2001 Aug;22(14):2908-35. doi: 10.1002/1522-2683(200108)22:14<2908::AID-ELPS2908>3.0.CO;2-M.
Proteomics relying on two-dimensional (2-D) gel electrophoresis of proteins followed by spot identification with mass spectrometry is an excellent experimental tool for physiological studies opening a new perspective for understanding overall cell physiology. This is the intriguing outcome of a method introduced by Klose and O'Farrell independently 25 years ago. Physiological proteomics requires a 2-D reference map on which most of the main proteins were identified. In this paper, we present such a reference map with more than 300 entries for Bacillus subtilis proteins with an isoelectric point (pI) between 4 and 7. The most abundant proteins of exponentially growing cells were compiled and shown to perform mainly housekeeping functions in glycolysis, tricarboxylic acid cycle (TCC), amino acid biosynthesis and translation as well as protein quality control. Furthermore, putative post-translational modifications were shown at a large scale, with 47 proteins in total forming more than one spot. In a few selected cases evidence for phosphorylation of these proteins is presented. The proteome analysis in the standard pI range was complemented by either stretching the most crowded regions in a narrow pH gradient 4.5-5.5, or by adding other fractions of the total B. subtilis proteome such as alkaline proteins as well as extracellular proteins. A big challenge for future studies is to provide an experimental protocol covering the fraction of intrinsic membrane proteins that almost totally escaped detection by the experimental procedure used in this study.
蛋白质组学依靠蛋白质的二维（2-D）凝胶电泳，随后通过质谱进行斑点鉴定，是生理研究的一种出色实验工具，为理解细胞整体生理学开辟了新视角。这是克洛泽（Klose）和奥法雷尔（O'Farrell）25年前独立引入的一种方法的有趣成果。生理蛋白质组学需要一张二维参考图谱，上面鉴定出了大多数主要蛋白质。在本文中，我们展示了这样一张参考图谱，其中包含300多个枯草芽孢杆菌蛋白质条目，其等电点（pI）在4到7之间。对数生长期细胞中最丰富的蛋白质被汇编在一起，结果表明它们主要在糖酵解、三羧酸循环（TCC）、氨基酸生物合成、翻译以及蛋白质质量控制中发挥看家功能。此外，大规模展示了假定的翻译后修饰，共有47种蛋白质形成了不止一个斑点。在少数选定案例中，给出了这些蛋白质磷酸化的证据。通过在狭窄的pH梯度4.5 - 5.5中扩展最拥挤区域，或者通过添加枯草芽孢杆菌总蛋白质组的其他部分，如碱性蛋白质和细胞外蛋白质，对标准pI范围内的蛋白质组分析进行了补充。未来研究面临的一个重大挑战是提供一个实验方案，以涵盖几乎完全逃脱本研究中使用的实验程序检测的内在膜蛋白部分。
