Cohen A M, Rumpel K, Coombs G H, Wastling J M
Division of Infection & Immunity, Joseph Black Building, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
Int J Parasitol. 2002 Jan;32(1):39-51. doi: 10.1016/s0020-7519(01)00308-3.
The development of tools for the analysis of global gene expression is vital for the optimal exploitation of the data on parasite genomes that are now being generated in abundance. Recent advances in two-dimensional electrophoresis (2-DE), mass spectrometry and bioinformatics have greatly enhanced the possibilities for mapping and characterisation of protein populations. We have employed these developments in a proteomics approach for the analysis of proteins expressed in the tachyzoite stage of Toxoplasma gondii. Over 1000 polypeptides were reproducibly separated by high-resolution 2-DE using the pH ranges 4-7 and 6-11. Further separations using narrow range gels suggest that at least 3000-4000 polypeptides should be resolvable by 2-DE using multiple single pH unit gels. Mass spectrometry was used to characterise a variety of protein spots on the 2-DE gels. Peptide mass fingerprints, acquired by matrix-assisted laser desorption/ionisation-(MALDI) mass spectrometry, enabled unambiguous protein identifications to be made where full gene sequence information was available. However, interpretation of peptide mass fingerprint data using the T. gondii expressed sequence tag (EST) database was less reliable. Peptide fragmentation data, acquired by post-source decay mass spectrometry, proved a more successful strategy for the putative identification of proteins using the T. gondii EST database and protein databases from other organisms. In some instances, several protein spots appeared to be encoded by the same gene, indicating that post-translational modification and/or alternative splicing events may be a common feature of functional gene expression in T. gondii. The data demonstrate that proteomic analyses are now viable for T. gondii and other protozoa for which there are good EST databases, even in the absence of complete genome sequence. Moreover, proteomics is of great value in interpreting and annotating EST databases.
开发用于分析全球基因表达的工具对于充分利用目前大量生成的寄生虫基因组数据至关重要。二维电泳（2-DE）、质谱和生物信息学的最新进展极大地增加了绘制和表征蛋白质群体的可能性。我们已将这些进展应用于蛋白质组学方法，以分析刚地弓形虫速殖子阶段表达的蛋白质。使用pH范围为4-7和6-11的高分辨率2-DE可重复分离出1000多种多肽。使用窄范围凝胶的进一步分离表明，使用多个单pH单位凝胶通过2-DE至少可分辨3000-4000种多肽。质谱用于表征2-DE凝胶上的各种蛋白质斑点。通过基质辅助激光解吸/电离-（MALDI）质谱获得的肽质量指纹图谱，在有完整基因序列信息的情况下能够明确鉴定蛋白质。然而，使用刚地弓形虫表达序列标签（EST）数据库解释肽质量指纹数据的可靠性较低。通过源后衰变质谱获得的肽片段化数据被证明是使用刚地弓形虫EST数据库和其他生物体的蛋白质数据库推定鉴定蛋白质的更成功策略。在某些情况下，几个蛋白质斑点似乎由同一基因编码，这表明翻译后修饰和/或可变剪接事件可能是刚地弓形虫功能基因表达的共同特征。数据表明，即使没有完整的基因组序列，蛋白质组学分析对于有良好EST数据库的刚地弓形虫和其他原生动物现在也是可行的。此外，蛋白质组学在解释和注释EST数据库方面具有重要价值。
