School of Molecular Bioscience, The University of Sydney, Australia.
Proteomics. 2011 Aug;11(15):3169-89. doi: 10.1002/pmic.201000808. Epub 2011 Jul 4.
The study of surface-exposed proteins has received increasing attention following the advent of genomic sequencing, which in turn has enabled predictive tools and facilitated the technologies for their analysis by proteomics. The exterior topology of a bacterial pathogen is the interface between the cell and environment and thus is the initial mediator for infection, providing an important reservoir for components that may be used for novel vaccine development as well as the characterization of new drug targets. The study of such biological molecules has however, been under-represented in proteomics studies due to the difficulty involved in their analysis. Cell-envelope proteins in bacteria are typically difficult to characterize due to their low abundance, poor solubility, and the problematic isolation of pure surface fractions with only minimal contamination. Here, we describe different cell envelope preparations for proteomic characterization, focused principally on gel-free technologies. Fractionation techniques popularly used in proteomics are also explained with emphasis on surface and membrane-derived proteins/peptides. Conditional confirmation of localization is also explored with emphasis on different prediction algorithms as well as on analyses of surface peptide fractions by the use of different search programs and their implications for the unambiguous identification of surface-exposed and membrane-embedded proteins. Finally, different quantification techniques are discussed that are important for the validation of identifications and for highlighting novel proteins that may warrant further study by independent techniques.
随着基因组测序的出现，对表面暴露蛋白的研究受到了越来越多的关注，这反过来又为预测工具提供了支持，并促进了通过蛋白质组学对其进行分析的技术。细菌病原体的外表面拓扑结构是细胞与环境之间的界面，因此是感染的最初介质，为可能用于新型疫苗开发以及新药物靶点表征的成分提供了重要的来源。然而，由于其分析的难度，这些生物分子的研究在蛋白质组学研究中一直没有得到充分体现。由于细菌细胞包膜蛋白丰度低、溶解度差，以及难以分离出只有最小污染的纯表面部分，因此它们通常难以进行表征。在这里，我们描述了不同的细胞包膜制备方法，用于蛋白质组学的特征分析，主要集中在无胶技术上。还解释了蛋白质组学中常用的分级技术，重点是表面和膜衍生的蛋白质/肽。还探讨了定位的条件确认，重点是不同的预测算法，以及通过使用不同的搜索程序对表面肽部分进行分析，以及它们对明确鉴定表面暴露和膜嵌入蛋白的影响。最后，讨论了不同的定量技术，这些技术对于鉴定的验证以及突出可能需要进一步通过独立技术进行研究的新型蛋白质非常重要。
