Wasinger V C, Cordwell S J, Cerpa-Poljak A, Yan J X, Gooley A A, Wilkins M R, Duncan M W, Harris R, Williams K L, Humphery-Smith I
Department of Microbiology, University of Sydney, Australia.
Electrophoresis. 1995 Jul;16(7):1090-4. doi: 10.1002/elps.11501601185.
A protein map of the smallest known self-replicating organism, Mycoplasma genitalium (Class: Mollicutes), revealed a high proportion of acidic proteins. Amino acid composition was used to putatively identify, or provide unique parameters, for 50 gene products separated by two-dimensional gel electrophoresis. A further 19 proteins were subjected to peptide-mass fingerprinting using matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry and 4 were subjected to N-terminal Edman degradation. The majority of M. genitalium proteins remain uncharacterised. However, the combined approach of amino acid analysis and peptide-mass fingerprinting allowed gene products to be linked to homologous genes in a variety of organisms. This has allowed proteins to be identified prior to detection of their respective genes via the M. genitalium sequencing initiative. The principle of 'hierarchical' analysis for the mass screening of proteins and the analysis of microbial genomes via their protein complement or 'proteome' is detailed. Here, characterisation of gene products depends upon the quickest and most economical technologies being employed initially, so as to determine if a large number of proteins are already present in both homologous and heterologous species databases. Initial screening, which lends itself to automation and robotics, can then be followed by more time and cost intensive procedures, when necessary.
已知最小的自我复制生物体——生殖支原体（类别：柔膜菌纲）的蛋白质图谱显示，酸性蛋白质占比很高。通过氨基酸组成，对二维凝胶电泳分离出的50种基因产物进行了推定鉴定，或提供了独特参数。另外19种蛋白质采用基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱进行肽质量指纹分析，4种蛋白质进行了N端埃德曼降解分析。大多数生殖支原体蛋白质仍未得到表征。然而，氨基酸分析和肽质量指纹分析相结合的方法，使得基因产物能够与多种生物体中的同源基因建立联系。这使得在通过生殖支原体测序计划检测到各自基因之前，就能鉴定出蛋白质。文中详细阐述了蛋白质大规模筛选的“分层”分析原理，以及通过微生物基因组的蛋白质互补物或“蛋白质组”对微生物基因组进行分析的原理。在此，基因产物的表征首先取决于采用最快且最经济的技术，以便确定同源和异源物种数据库中是否已经存在大量蛋白质。初步筛选适合自动化和机器人操作，如有必要，随后可采用更耗时且成本更高的程序。
