Analysis of intracellular short organic acid-coenzyme A esters from actinomycetes using liquid chromatography-electrospray ionization-mass spectrometry

✍ Scribed by Je Won Park; Won Seok Jung; Sung Ryeol Park; Byoung Chul Park; Yeo Joon Yoon

• Publisher: John Wiley and Sons
• Year: 2007
• Tongue: English
• Weight: 334 KB
• Volume: 42
• Category: Article
• ISSN: 1076-5174
• DOI: 10.1002/jms.1240

✦ Synopsis

Abstract

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 © 2007 John Wiley & Sons, Ltd.