LC-quadrupole/Orbitrap high-resolution mass spectrometry enables stable isotope-resolved simultaneous quantification and ¹³C-isotopic labeling of acyl-coenzyme A thioesters

Abstract

Acyl-coenzyme A (acyl-CoA) thioesters are evolutionarily conserved, compartmentalized, and energetically activated substrates for biochemical reactions. The ubiquitous involvement of acyl-CoA thioesters 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-CoA thioesters. 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-CoA thioesters can be labeled with either a fixed [(13)C3(15)N1] label derived from pantothenate into the CoA moiety or via variable [(13)C] 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-CoA thioesters by stable isotope dilution using the [(13)C3(15)N1] label and measurement of the incorporation of labeled carbon atoms derived from [(13)C6]-glucose, [(13)C5(15)N2]-glutamine, and [(13)C3]-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-CoA thioesters.

Keywords: Bioanalytical methods; Coenzyme A; Mass spectrometry; Metabolism; Stable isotope labeling.

Figure 1

Metabolism, isotopic tracing, and mass spectral identification of acyl-CoAs. (A) Isotopic tracers incorporated into acyl-CoA species, including acetyl-, succinyl-, and propionyl-CoA. (B) Structure and fragmentation of acyl-CoAs.

Figure 2

Theoretical and experimental resolution of M4 isotopologue of succinyl-CoA demonstrates that both tandem MS and ultra-high resolution are required for stable isotope resolution of acyl-CoAs. Theoretical Gaussian resolution (FWHM) with 15 points per sample are shown for 280000,140000, 70000, 35000, 17500 resolution, predicted centroid masses at each resolution are indicated on the graph for (A) the [M+H]⁺ of succinyl-CoA and (B) the [M-507+H]⁺ fragment ion of succinyl-CoA and (C) a 1:1 complex mixture of ¹³C₄ and ¹³C₃¹⁵N₁ labeled succinyl-CoA. (D) Experimentally determined resolutions [R] by LC-HRMS (broken blue line) and LC-MS/HRMS (solid black line) of the M4 isotopologue from cell extract of DLCL2 cells grown with ¹³C₅¹⁵N₂ glutamine for (top) [M+H]⁺ of succinyl-CoA and (bottom) the [M-507+H]⁺ fragment ion of succinyl-CoA. (E) LC-HRMS incompletely resolves the ¹³C₄ and ¹³C₃¹⁵N₁ labels, resulting in an erroneous quantification of the ¹³C₄ label. (F) LC-MS/HRMS completely resolves the ¹³C₄ and ¹³C₃¹⁵N₁ labels correctly quantifying the isotopologue distribution. Chromatograms have been offset for clarity.