Nontargeted Profiling of Coenzyme A thioesters in biological samples by tandem mass spectrometry

Abstract

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.

Organ-Specific Cancer Metabolism and Its Potential for Therapy

“…Moreover, it was shown that Stat3-mediated activation of the microRNA23a suppresses gluconeogenesis by targeting glucose-6-phosphatase expression [79]. Consistently, restoring gluconeogenesis by dexamethasone treatment showed significant inhibition of in vivo tumor growth [80]. A block in gluconeogenesis might contribute to the survival of HCC cells by increased usage of glycolysis and pentose phosphate pathway due to accumulation of glucose-6-phosphate [79].…”

“…Ma et al showed in 2013 that this lack of gluconeogenesis might occur due to the decreased expression of 11β-hydroxysteroid dehydrogenase type 1 and an increased expression of 11β-hydroxysteroid dehydrogenase type 2. These enzymes control the activity of glucocorticoids [80]. Glucocorticoids promote gluconeogenesis, but in liver cancer cells the altered expression of 11β-hydroxysteroid dehydrogenase1/2 results in the insensitiveness of liver cancer cells to endogenous glucocorticoids [81].…”

High-Resolution Metabolomics with Acyl-CoA Profiling Reveals Widespread Remodeling in Response to Diet*

“…Optima ammonium acetate, ammonium hydroxide, Optima LC-MS grade formic acid, acetonitrile, methanol, and water were purchased from Fisher Scientific, Agawam, MA. U- 13 Mice were fasted for 4 h before they were sacrificed by cervical dislocation and tissues were snap frozen in liquid nitrogen. Frozen tissues were stored at Ϫ80°C for further analysis (23).…”

“…Preparation of 13 C Labeled Extract and Evaluation of Tissue Extraction Method-Escherichia coli strain NCM3722 (E. coli) was a generous gift from Joshua Rabinowitz's group (Princeton University). The E. coli culture and metabolite extraction method were modified from a previous studies (25,26).…”

“…However, these methods involve several laborious steps of sample purification and enrichment before LC-MS analysis, such as solid phase extraction, which in addition to often being time-and cost-prohibitive, can also result in poor sensitivity and accuracy because of imperfect metabolite recovery. Moreover, reversed phase ion-paired chromatogra-phy coupled to high-resolution MS has also been used for short, medium, and long chain acyl-CoA identification or quantification with the help of stable isotope labeled standards (10,13). However, these methods were also developed with limited coverage of metabolites, and the quantitative capacity without using stable isotope labeled standards was not evaluated.…”