Institute for Biotechnology and Bioengineering, Biological Sciences Research Group, Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Technical University of Lisbon, Av. Rovisco Pais, Lisbon, Portugal.
Expert Rev Proteomics. 2010 Dec;7(6):943-53. doi: 10.1586/epr.10.76.
Quantitative proteomics based on 2D electrophoresis (2-DE) coupled with peptide mass fingerprinting is still one of the most widely used quantitative proteomics approaches in microbiology research. Our view on the exploitation of this global expression analysis technique and its contribution and potential to push forward the field of molecular microbial physiology towards a molecular systems microbiology perspective is discussed in this article. The advances registered in 2-DE-based quantitative proteomic analysis leading to increased protein resolution, sensitivity and accuracy, and the promising use of 2-DE to gain insights into post-translational modifications at a proteome-wide level (considering all the proteins/protein forms expressed by the genome) are focused on. Given the progress made in this field, it is foreseen that the 2-DE-based approach to quantitative proteomics will continue to be a fundamental tool for microbiologists working at a genome-wide scale. Guidelines are also provided for the exploitation of expression proteomics data, based on useful computational tools, and for the integration of these data with other genome-wide expression information. The advantages and limitations of a complete 2-DE-based expression proteomics analysis, envisaging the quantification of the global changes occurring in the proteome of a given cell depending on environmental or genetic manipulations, are discussed from the microbiologist's perspective. Particular focus is given to the emerging field of toxicoproteomics, a new systems toxicity approach that offers a powerful tool to directly monitor the earliest stages of the toxicological response by identifying critical proteins and pathways that are affected by, and respond to, a chemical stress. The experimental design and the bioinformatics analysis of data used in our laboratory to gain mechanistic insights through expression proteomics into the responses of the eukaryotic model Saccharomyces cerevisiae or of Pseudomonas strains to environmental toxicants are presented as case studies.
基于二维电泳（2-DE）结合肽质量指纹图谱的定量蛋白质组学仍然是微生物学研究中最广泛使用的定量蛋白质组学方法之一。本文讨论了我们对这种全局表达分析技术的开发及其对推动分子微生物生理学领域向分子系统微生物学观点发展的贡献和潜力的看法。本文重点介绍了基于 2-DE 的定量蛋白质组学分析在提高蛋白质分辨率、灵敏度和准确性方面取得的进展，以及 2-DE 在获得全蛋白质组水平（考虑基因组表达的所有蛋白质/蛋白质形式）的翻译后修饰方面的有希望的应用。考虑到该领域的进展，可以预见，基于 2-DE 的定量蛋白质组学方法将继续成为从事全基因组规模工作的微生物学家的基本工具。还提供了基于有用的计算工具的表达蛋白质组学数据的开发指南，以及将这些数据与其他全基因组表达信息集成的指南。从微生物学家的角度讨论了完整的基于 2-DE 的表达蛋白质组学分析的优缺点，设想根据环境或遗传操作来量化给定细胞中蛋白质组中发生的全局变化。特别关注毒理学蛋白质组学这一新兴领域，这是一种新的系统毒性方法，通过鉴定受化学应激影响并对其作出反应的关键蛋白质和途径，提供了直接监测毒理学反应早期阶段的强大工具。通过表达蛋白质组学获得真核模型酿酒酵母或假单胞菌菌株对环境毒物的响应的机制见解，介绍了我们实验室用于获得机制见解的实验设计和生物信息学数据分析作为案例研究。
