LC-MS/MS quantification of short-chain acyl-CoA’s in Escherichia coli demonstrates versatile propionyl-CoA synthetase substrate specificity

Armando, John; Boghigian, Brett A.; Pfeifer, Blaine A.

doi:10.1111/j.1472-765x.2011.03184.x

Cited by 35 publications

(31 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In order to match the specific chemical properties, it is inevitable to use CoAs as internal standards (ISs). Rare CoAs, such as propionyl-CoA or glutaryl-CoA, were used in the literature as IS for the analysis of acetyl-CoA or malonyl-CoA [19,12,11]. However, it is commonly accepted that the ideal internal standardization is provided by isotopically enriched standards.…”

Section: Validation Of Quantification Methodsmentioning

confidence: 99%

“…Reversed phase liquid chromatography employing ion-pairing reagents (ion-pair RPLC) was addressed as the method of choice suitable for a broad spectrum of short-chain acyl-CoA analysis [11,12,13,14,15]. Alternately, a comparatively smaller selection of these target metabolites has been separated by reversed phase liquid chromatography without the use of ion-pairing reagents [16,17,18,19]. In studies where short-chain acyl-CoAs were measured within a comprehensive primary metabolome analysis, ion-pair RPLC was applied [20,21].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

LC-MS/MS-based analysis of coenzyme A and short-chain acyl-coenzyme A thioesters

et al. 2015

View full text Add to dashboard Cite

Absolute quantification of intracellular coenzyme A (CoA), coenzyme A disulfide, and short-chain acyl-coenzyme A thioesters was addressed by developing a tailored metabolite profiling method based on liquid chromatography in combination with tandem mass spectrometric detection (LC-MS/MS). A reversed phase chromatographic separation was established which is capable of separating a broad spectrum of CoA, its corresponding derivatives, and their isomers despite the fact that no ion-pairing reagent was used (which was considered as a key advantage of the method). Excellent analytical figures of merit such as high sensitivity (LODs in the nM to sub-nM range) and high repeatability (routinely 4 %; N = 15) were obtained. Method validation comprised a study on standard purity, stability, and recoveries during sample preparation. Uniformly labeled U(13)C yeast cell extracts offered ideal internal standards for validation purposes and for a quantification exercise in the rumen bacterium Megasphaera elsdenii.

show abstract

Section: Validation Of Quantification Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

LC-MS/MS-based analysis of coenzyme A and short-chain acyl-coenzyme A thioesters

et al. 2015

View full text Add to dashboard Cite

show abstract

“…The resuspended extracts were incubated on ice with intermittent vortexing for 15 min. An equal molar volume of ammonium hydroxide was added postincubation to neutralize the acetic acid, and each extract was centrifuged for 3 min at 15,700 ϫ g, transferred to a new 1.5-ml microcentrifuge tube, and centrifuged for 5 min at 15,700 ϫ g. The clarified extract (10 l) was injected for HPLC-MS/MS analysis (46,47).…”

Section: Methodsmentioning

confidence: 99%

Loss of Cardiolipin Leads to Perturbation of Acetyl-CoA Synthesis

Raja

Joshi

et al. 2017

Journal of Biological Chemistry

View full text Add to dashboard Cite

Edited by George M. CarmanCardiolipin (CL), the signature phospholipid of mitochondrial membranes, plays an important role in mitochondrial processes and bioenergetics. CL is synthesized de novo and undergoes remodeling in the mitochondrial membranes. Perturbation of CL remodeling leads to the rare X-linked genetic disorder Barth syndrome, which shows disparities in clinical presentation. To uncover biochemical modifiers that exacerbate CL deficiency, we carried out a synthetic genetic array screen to identify synthetic lethal interactions with the yeast CL synthase mutant crd1⌬. The results indicated that crd1⌬ is synthetically lethal with mutants in pyruvate dehydrogenase (PDH), which catalyzes the conversion of pyruvate to acetyl-CoA. Acetyl-CoA levels were decreased in the mutant. The synthesis of acetylCoA depends primarily on the PDH-catalyzed conversion of pyruvate in the mitochondria and on the PDH bypass in the cytosol, which synthesizes acetyl-CoA from acetate. Consistent with perturbation of the PDH bypass, crd1⌬ cells grown on acetate as the sole carbon source exhibited decreased growth, decreased acetyl-CoA, and increased intracellular acetate levels resulting from decreased acetyl-CoA synthetase activity. PDH mRNA and protein levels were up-regulated in crd1⌬ cells, but PDH enzyme activity was not increased, indicating that PDH up-regulation did not compensate for defects in the PDH bypass. These findings demonstrate for the first time that CL is required for acetyl-CoA synthesis, which is decreased in CL-deficient cells as a result of a defective PDH bypass pathway. Cardiolipin (CL)4 is a unique phospholipid with dimeric structure that constitutes about 15% of the total phospholipid in mitochondria (1-4). CL is synthesized de novo in the inner mitochondrial membrane (5) and undergoes remodeling in which saturated fatty acyl chains are replaced with unsaturated fatty acids (6, 7). CL plays an important role in maintenance of mitochondrial structure, interaction with mitochondrial membrane proteins, respiration and stability of respiratory chain supercomplexes (8 -10), and other mitochondrial functions such as protein import and maintenance of the membrane potential (11-13). Loss of CL perturbs mitochondrial bioenergetics and decreases ATP synthesis (12, 14 -16). Aberrant CL remodeling due to mutation of tafazzin, the transacylase that remodels CL, leads to the severe genetic disorder Barth syndrome (BTHS) (17). Loss of tafazzin results in decreased total CL, increased monolysocardiolipin, and aberrant CL species (18 -20). In yeast, tafazzin mutant phenotypes are not due to aberrant CL species but more likely result from decreased total CL/increased monolysocardiolipin (21, 22). The clinical presentation of BTHS includes cardio-and skeletal myopathy, neutropenia, 3-methylglutaconic aciduria, growth retardation, abnormal mitochondria, and defective oxidative phosphorylation (23). However, disparities in the clinical manifestation are characteristic of the disorder (24 -26), indicating that physiologi...

show abstract

“…However, most methods only assay either short-, medium-, or long-chain acyl-CoAs (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). The best approach for acyl-CoA quantitation, so far, is to measure the target acyl-CoAs from short to long carbon chain length in a single method ( 20,21 ).…”

Section: Lc-ms/ms Is the Most Sensitive Methods To Analyze Acylcoas Anmentioning

confidence: 99%

Novel approach in LC-MS/MS using MRM to generate a full profile of acyl-CoAs: discovery of acyl-dephospho-CoAs

Zhang

Berthiaume

et al. 2014

Journal of Lipid Research

View full text Add to dashboard Cite

This article is available online at http://www.jlr.org Acyl-CoAs are a class of important molecules that play essential roles in many physiological processes ( 1 ), such as fatty acid oxidation, lipid synthesis/remodeling, ketone body synthesis, xenobiotic metabolism, and signaling pathways. Classically, acyl-CoAs such as free CoA, acetyl-CoA, and malonyl-CoA are recognized as regulators of metabolic fl ux. The ratio of acetyl-CoA versus free CoA tightly regulates glycolysis and fatty acid oxidation. Malonyl-CoA attenuates fatty acid oxidation by inhibiting acyl-CoA transport into the mitochondrion for oxidation and is utilized in fatty acid synthesis when its concentration is elevated ( 2 ). Many proteins and genes are dynamically regulated by deacylation and acylation via various acyl-CoAs, such as acetyl-CoA, succinyl-CoA, palmitoyl-CoA, etc. ( 3 ). However, acyl-CoAs present in the cell are diverse and may involve molecules beyond fatty acids and their oxidized derivatives. The metabolism of xenobiotics can also lead to the formation of acyl-CoAs, as demonstrated in our previous work on the metabolism of 4-hydroxy acids from C 4 to C 11 in perfused rat liver. The identifi cation of these novel acyl-CoAs extends our understanding of the new catabolic pathways involved in the disposal of 4-hydroxy acids including drugs of abuse and lipid peroxidation products ( 4-8 ).Acyl-CoAs are exclusive to the intracellular metabolites, and the profi le of these biomolecules is indicative of the local metabolic status. Each organ has its specifi c physiological roles with different energetic demands, and therefore, would be expected to exhibit a unique acyl-CoA profi le. The heart preferentially utilizes fatty acids to meet the ATP demand of mechanical contraction. Glucose and/or ketone bodies serve as the primary substrate of the brain for energy

show abstract

LC-MS/MS quantification of short-chain acyl-CoA’s in Escherichia coli demonstrates versatile propionyl-CoA synthetase substrate specificity

Cited by 35 publications

References 20 publications

LC-MS/MS-based analysis of coenzyme A and short-chain acyl-coenzyme A thioesters

LC-MS/MS-based analysis of coenzyme A and short-chain acyl-coenzyme A thioesters

Loss of Cardiolipin Leads to Perturbation of Acetyl-CoA Synthesis

Novel approach in LC-MS/MS using MRM to generate a full profile of acyl-CoAs: discovery of acyl-dephospho-CoAs

Contact Info

Product

Resources

About