LC-quadrupole/Orbitrap high-resolution mass spectrometry enables stable isotope-resolved simultaneous quantification and 13C-isotopic labeling of acyl-coenzyme A thioesters
Abstract:Acyl-coenyzme A thioesters (acyl-CoAs) are evolutionarily conserved, compartmentalized, and energetically activated substrates for biochemical reactions. The ubiquitous involvement of acyl-CoAs in metabolism, including the tricarboxylic acid cycle, fatty acid metabolism, amino acid degradation, and cholesterol metabolism highlights the broad applicability of applied measurements of acyl-CoAs. However, quantitation of acyl-CoA levels provides only one dimension of metabolic information and a more complete descr… Show more
“…For quantitation, internal standards containing [ 13 C 3 15 N 1 ]-labelled acyl-CoAs generated in pan6 -deficient yeast culture (Snyder et al, 2015) were added to each sample in equal amounts. Samples were analyzed by an Ultimate 3000 autosampler coupled to a Thermo Q Exactive Plus instrument in positive ESI mode using the settings described previously (Frey et al, 2016). A more detailed method can be found in the Supplemental Material.…”
SUMMARY
Mechanisms of metabolic flexibility enable cells to survive under stressful conditions and can thwart therapeutic responses. Acetyl-CoA plays central roles in energy production, lipid metabolism, and epigenomic modifications. Here we show that upon genetic deletion of Acly, the gene coding for ATP-citrate lyase (ACLY), cells remain viable and proliferate, although at an impaired rate. In the absence of ACLY, cells upregulate ACSS2 and utilize exogenous acetate to provide acetyl-CoA for de novo lipogenesis (DNL) and histone acetylation. A physiological level of acetate is sufficient for cell viability and abundant acetyl-CoA production, although histone acetylation levels remain low in ACLY-deficient cells unless supplemented with high levels of acetate. ACLY-deficient adipocytes accumulate lipid in vivo, exhibit increased acetyl-CoA and malonyl-CoA production from acetate, and display some differences in fatty acid content and synthesis. Together, these data indicate that engagement of acetate metabolism is a crucial, although partial, mechanism of compensation for ACLY deficiency.
“…For quantitation, internal standards containing [ 13 C 3 15 N 1 ]-labelled acyl-CoAs generated in pan6 -deficient yeast culture (Snyder et al, 2015) were added to each sample in equal amounts. Samples were analyzed by an Ultimate 3000 autosampler coupled to a Thermo Q Exactive Plus instrument in positive ESI mode using the settings described previously (Frey et al, 2016). A more detailed method can be found in the Supplemental Material.…”
SUMMARY
Mechanisms of metabolic flexibility enable cells to survive under stressful conditions and can thwart therapeutic responses. Acetyl-CoA plays central roles in energy production, lipid metabolism, and epigenomic modifications. Here we show that upon genetic deletion of Acly, the gene coding for ATP-citrate lyase (ACLY), cells remain viable and proliferate, although at an impaired rate. In the absence of ACLY, cells upregulate ACSS2 and utilize exogenous acetate to provide acetyl-CoA for de novo lipogenesis (DNL) and histone acetylation. A physiological level of acetate is sufficient for cell viability and abundant acetyl-CoA production, although histone acetylation levels remain low in ACLY-deficient cells unless supplemented with high levels of acetate. ACLY-deficient adipocytes accumulate lipid in vivo, exhibit increased acetyl-CoA and malonyl-CoA production from acetate, and display some differences in fatty acid content and synthesis. Together, these data indicate that engagement of acetate metabolism is a crucial, although partial, mechanism of compensation for ACLY deficiency.
“…Liquid Chromatography-Mass Spectrometry-Samples were analyzed by an Ultimate 3000 autosampler coupled to a Thermo Q Exactive Plus instrument in positive electrospray ionization mode in 10-l injections using settings described previously (41). Analytes were separated before introduction to the mass spectrometer using a Waters HSS T3 column (2.1 ϫ 100 mm, 3.5-m particle size) with 5 mM ammonium acetate in water as solvent A, 5 mM ammonium acetate in acetonitrile/ water (95:5, v/v) as solvent B, and acetonitrile/water/formic acid (80:20:0.1, v/v/v) as solvent C. Gradient conditions were as follows: 2% B for 1.5 min, increased to 25% over 3.5 min, increased to 100% B in 0.5 min and held for 8.5 min, and washed with 100% C for 5 min before equilibration for 5 min.…”
Section: Determination Of Acyl-coa Speciesmentioning
Edited by John M. DenuCellular metabolism dynamically regulates the epigenome via availability of the metabolite substrates of chromatin-modifying enzymes. The impact of diet on the metabolism-epigenome axis is poorly understood but could alter gene expression and influence metabolic health. ATP citrate-lyase produces acetyl-CoA in the nucleus and cytosol and regulates histone acetylation levels in many cell types. Consumption of a high-fat diet (HFD) results in suppression of ATP citrate-lyase levels in tissues such as adipose and liver, but the impact of diet on acetyl-CoA and histone acetylation in these tissues remains unknown. Here we examined the effects of HFD on levels of acyl-CoAs and histone acetylation in mouse white adipose tissue (WAT), liver, and pancreas. We report that mice consuming a HFD have reduced levels of acetyl-CoA and/or acetyl-CoA:CoA ratio in these tissues. In WAT and the pancreas, HFD also impacted the levels of histone acetylation; in particular, histone H3 lysine 23 acetylation was lower in HFD-fed mice. Genetic deletion of Acly in cultured adipocytes also suppressed acetyl-CoA and histone acetylation levels. In the liver, no significant effects on histone acetylation were observed with a HFD despite lower acetyl-CoA levels. Intriguingly, acetylation of several histone lysines correlated with the acetyl-CoA: (iso)butyryl-CoA ratio in liver. ButyrylCoA and isobutyryl-CoA interacted with the acetyltransferase P300/CBP-associated factor (PCAF) in liver lysates and inhibited its activity in vitro. This study thus provides evidence that diet can impact tissue acyl-CoA and histone acetylation levels and that acetyl-CoA abundance correlates with acetylation of specific histone lysines in WAT but not in the liver.
“…Instruments combining HR/AM MS capabilities with more traditional isolation and fragmentation capabilities, including the quadrupole/Orbitrap or quadrupole/time-of-flight (TOF) hybrid instruments, can provide sensitive, specific quantification through multiple modes of mass spectrometry at scan speeds increasingly compatible with liquid chromatography. In hybrid instruments equipped with a mass filter to select precursor ions, a fragmentation cell, and then a high resolution mass analyzer, MS/HRMS analysis can also be performed [14]. The product ion chromatograms can be extracted from the MS/high-resolution MS data using a narrow mass tolerance (<5 ppm) commensurate with the mass accuracy of the analyzer, the resolution of the instrument and the complexity of the matrix.…”
A multiplexed quantitative method for the analysis of three major unconjugated steroids in human serum by stable isotope dilution liquid chromatography-high resolution mass spectrometry (LC-HRMS) was developed and validated on a Q. Exactive Plus hybrid quadrupole/Orbitrap mass spectrometer. This quantification utilized isotope dilution and Girard P derivatization on the keto-groups of testosterone (T), androstenedione (AD) and dehydroepiandrosterone (DHEA) to improve ionization efficiency using electrospray ionization. Major isomeric compounds to T and DHEA; the inactive epimer of testosterone (epiT), and the metabolite of AD, 5α-androstanedione (5α-AD) were completely resolved on a biphenyl column within an 18 min method. Inter- and intra-day method validation using LC-HRMS with qualifying product ions was performed and acceptable analytical performance was achieved. The method was further validated by comparing steroid levels from 100 μL of serum from young vs older subjects. Since this approach provides high-dimensional HRMS data, untargeted analysis by age group was performed. DHEA and T were detected among the top analytes most significantly different across the two groups after untargeted LC-HRMS analysis, as well as a number of other still unknown metabolites, indicating the potential for combined targeted/untargeted analysis in steroid analysis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.