Singh Om V, Chandel Anuj K
Division of Biological and Health Science, University of Pittsburgh-Bradford, Bradford, PA 16701, USA.
Methods Mol Biol. 2010;599:141-56. doi: 10.1007/978-1-60761-439-5_10.
Environmental contamination has been viewed as an ecological malaise for which bioremediation can be prescribed as a "perfect medicine." The solution to the problems with bioremediation lies in analyzing to what extent the microbes' physiological machinery contributes to the degradation process and which biomolecules and their mechanisms are responsible for regulatory factors within the degradation system, such as protein, metabolite, and enzymatic chemical transformation. In the post-genomic era, recent advances in proteomics have allowed us to elucidate many complex biological mechanisms. Two-dimensional gel electrophoresis (2DE) in conjunction with mass spectrometry (MS) can be utilized to identify the biomolecules and their molecular mechanisms in bioremediation. A set of highly abundant global proteins over a pI range 4-7 was separated and compared by size fractionation (25-100 kDa) on 2DE. We identified a set of catabolic proteins, enzymes, and heat shock molecular chaperones associated with the regulatory network that was found to be overexpressed under phenol-stressed conditions. This chapter also offers optimized ideal directions for 2DE, followed by easy-to-follow directions for a protein identification strategy using MALDI-TOF and targeting novel proteins/enzymes for a universal set of experiments.
环境污染一直被视为一种生态弊病，对此生物修复可被视为一种“完美药物”。生物修复问题的解决方案在于分析微生物的生理机制在多大程度上有助于降解过程，以及哪些生物分子及其机制负责降解系统中的调控因素，如蛋白质、代谢物和酶促化学转化。在后基因组时代，蛋白质组学的最新进展使我们能够阐明许多复杂的生物学机制。二维凝胶电泳（2DE）结合质谱（MS）可用于识别生物修复中的生物分子及其分子机制。通过在2DE上进行大小分级（25-100 kDa），分离并比较了一组在4-7的pI范围内高度丰富的全局蛋白质。我们鉴定出一组与调控网络相关的分解代谢蛋白质、酶和热休克分子伴侣，发现在苯酚胁迫条件下这些物质会过度表达。本章还提供了2DE的优化理想方向，随后是使用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）进行蛋白质鉴定策略的易于遵循的方向，以及针对一组通用实验靶向新型蛋白质/酶的方向。
