期刊
ANALYTICAL CHEMISTRY
卷 85, 期 17, 页码 8284-8290出版社
AMER CHEMICAL SOC
DOI: 10.1021/ac401555n
关键词
-
资金
- EU FP7 project SysteMTb
Coenzyme A (CoA) thioesters are ubiquitously present in metabolic networks and play a pivotal role in enzymatic formation and cleavage of carbon carbon bonds. We present a method allowing nontargeted profiling of CoA-thioesters in biological samples. The reported UHPLC-MS/MS approach employes ion-pairing chromatography to separate CoA-metabolites carrying different chemical functionalities such as hydroxyl or multiple carboxyl groups and to distinguish between isomers. Selective detection of CoA-thioesters is accomplished by precursor ion scanning on a triple quadrupole mass spectrometer and takes advantage of the abundant fragment with m/z = -408 that originates from the CoA-moiety. We used a mixture of 19 commercially available CoA-derivatives to develop and optimize our method. As a proof of concept we demonstrated detection of the major CoA-intermediates of branched chain amino acid degradation in biological samples. We then applied our method to investigate degradation of lipids in the microorganism Mycobacterium smegmatis. Profiling of CoA-thioesters led to the discovery of a novel intermediate of cholesterol degradation. This demonstrates the power of our method for untargeted profiling of CoA-thioesters and adds a missing link in mycobacterial cholesterol catabolism.
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