Metaproteomic and metagenomic analyses of defined oceanic microbial populations using microwave cell fixation and flow cytometric sorting.
Metaproteomic and metagenomic analyses of defined oceanic microbial populations using microwave cell fixation and flow cytometric sorting.
复制标题
使用微波细胞固定和流式细胞术分选对确定的海洋微生物种群进行宏蛋白质组学和宏基因组分析。
作者：
I. Mary;Anna E. Oliver;P. Skipp;Ross J. Holland;J. Topping;G. Tarran;David J. Scanlan;C. D. O'Connor;Andrew S. Whiteley;P. Burkill;M. Zubkov
对天然微生物细胞，特别是海洋微生物细胞进行分子研究的一个主要障碍是如何将其充分保存，以便进一步在陆地上进行分子分析。在这里，我们研究了在高速流式细胞仪分选之前使用微波进行细胞固定，以确定关键微生物种群的元蛋白组和元基因组。利用聚球藻、光合真核生物微小单胞菌和γ-变形菌变异盐单胞菌的培养物建立了微波固定程序。鸟枪蛋白质组学分析表明，微波固定和未固定的聚球藻WH 8102细胞的概况是相同的，因此微波固定的分选细胞的蛋白质组鉴定的nanoLC-MS/MS是可能的。微波固定流式分选的聚球藻细胞也可以成功地用于全基因组扩增和fosmid文库构建。然后，我们进行了成功的元蛋白质组学和宏基因组学分析的微波固定聚球藻细胞流从浓缩的微生物细胞，收集在北大西洋。因此，微波固定程序开发似乎是有用的微生物种群在水生生态系统中的分子研究。
A major obstacle in the molecular investigation of natural, especially oceanic, microbial cells is their adequate preservation for further land-based molecular analyses. Here, we examined the use of microwaves for cell fixation before high-speed flow cytometric sorting to define the metaproteomes and metagenomes of key microbial populations. The microwave fixation procedure was established using cultures of Synechococcus cyanobacteria, the photosynthetic eukaryote Micromonas pusilla and the gammaproteobacterium Halomonas variabilis. Shotgun proteomic analyses showed that the profile of microwave-fixed and -unfixed Synechococcus sp. WH8102 cells was the same, and hence proteome identification of microwave-fixed sorted cells by nanoLC-MS/MS is possible. Microwave-fixed flow-sorted Synechococcus cells can also be successfully used for whole-genome amplification and fosmid library construction. We then carried out successful metaproteomic and metagenomic analyses of microwave-fixed Synechococcus cells flow sorted from concentrates of microbial cells, collected in the North Atlantic Ocean. Thus, the microwave fixation procedure developed appears to be useful for molecular studies of microbial populations in aquatic ecosystems.
DOI：
10.1073/pnas.160270797
发表时间：
2000-08-15
影响因子：
11.1
作者：
Gygi, SP;Corthals, GL;Aebersold, R
通讯作者：
Aebersold, R