Analysis of intracellular short organic acid-coenzyme A esters from actinomycetes using liquid chromatography-electrospray ionization-mass spectrometry
Analysis of intracellular short organic acid-coenzyme A esters from actinomycetes using liquid chromatography-electrospray ionization-mass spectrometry
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DOI：
10.1002/jms.1240
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发表时间：
2007-09-01
影响因子：
2.3
通讯作者：
Yoon, Yeo Joon
中科院分区：
文献类型：
--
作者：
Park, Je Won;Jung, Won Seok;Yoon, Yeo Joon
建立了一种硅油密度离心、固相萃取（SPE）净化和LC-ESI-MS/MS分析的方法，用于快速、选择性、灵敏度和定量检测放线菌胞内短有机酸-辅酶A酯。采用LC-ESI-MS/MS方法分析的每个标准辅酶a -酯的检出限约为0.8 pmol （1.2 ng/ml）。对coa -酯特异性的典型片段离子（m/z 428）的选择离子色谱可以检测到参与初级和次级代谢的8种细胞内coa -酯。通过分析产生多酮类抗生素（甲霉素和匹克霉素）的委内瑞拉链霉菌野生型菌株、聚酮合成酶（PKS）缺失的委内瑞拉链霉菌突变体和表达外源PKS基因的委内瑞拉链霉菌突变体的细胞内辅酶a酯池，证明了该方法在细菌代谢组学研究中的应用。通过对委内瑞拉葡萄球菌3种不同基因型的coa -酯水平进行定量分析，可以进一步了解coa -酯在聚酮生物合成中的作用。这种分析方法可以扩展到各种微生物体内coa -酯池的大小和组成的定量，并可以详细了解体内coa -酯池与药用重要聚酮生产之间的关系。版权所有(c) 2007约翰威利父子有限公司
A method employing silicone oil density centrifugation, solid-phase extraction (SPE) cleanup, and LC-ESI-MS/MS analysis was developed for the rapid, selective, sensitive, and quantitative detection of an intracellular pool of short organic acid-CoA esters in actinomycetes. The detection limit was determined to be approximately 0.8 pmol (1.2 ng/ml) for each standard CoA-ester analyzed by the present LC-ESI-MS/MS method. A selected ion chromatogram for a typical fragment ion (m/z 428) specific to CoA-esters enabled the detection of eight intracellular CoA-esters involved in both primary and secondary metabolisms. The application of this method to bacterial metabolomic study is demonstrated by the profiling of the intracellular CoA-ester pools in the wild-type Streptomyces venezuelae strain producing polyketide antibiotics (methymycin and pikromycin), a polyketide synthase (PKS)-deleted S. venezuelae mutant, and a S. venezuelae mutant expressing the heterologous PKS genes. By quantifying the individual CoA-esterlevel in three different genotypes of the S. venezuela e strain, further insight could be gained into the role of CoA-estersin polyketide biosynthesis. This analytical approach can be extended to the quantification of the size and composition of in vivo CoA-ester pools in various microbes, and can provide a detailed understanding of the relationship between the in vivo CoA-ester pool and the production of pharmaceutically important polyketides. Copyright (c) 2007 John Wiley & Sons, Ltd.