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 description of metabolism requires the relative contribution of different precursors to individual substrates and pathways. Using two distinct stable isotope labeling approaches, acyl-CoAs can be labeled with either a fixed [13C315N1] label derived from pantothenate into the CoA moiety, or via variable [13C] labeling into the acyl-chain from metabolic precursors. Liquid chromatography-hybrid quadrupole/Orbitrap high resolution mass spectrometry using parallel reaction monitoring, but not single ion monitoring, allowed the simultaneous quantitation of acyl-CoAs by stable isotope dilution using the [13C315N1] label and measurement of the incorporation of labeled carbon atoms derived from [13C6]-glucose, [13C515N2]-glutamine and [13C3]-propionate. As a proof-of-principle we applied this method to human B-cell lymphoma (WSU-DLCL2) cells in culture, to precisely describe the relative pool size and enrichment of isotopic tracers into acetyl-, succinyl-, and propionyl-CoA. This method will allow highly precise, multiplexed, and stable isotope resolved determination of metabolism to refine metabolic models, characterize novel metabolism, and test modulators of metabolic pathways involving acyl-CoAs.

ATP-Citrate Lyase Controls a Glucose-to-Acetate Metabolic Switch

“…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.…”

Impact of a High-fat Diet on Tissue Acyl-CoA and Histone Acetylation Levels

“…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.…”

Validation of highly sensitive simultaneous targeted and untargeted analysis of keto-steroids by Girard P derivatization and stable isotope dilution-liquid chromatography-high resolution mass spectrometry

“…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.…”