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

• Published in: Analytical and bioanalytical chemistry Vol. 408; no. 13; p. 3651
• Main Authors: Frey, Alexander J, Feldman, Daniel R, Trefely, Sophie, Worth, Andrew J, Basu, Sankha S, Snyder, Nathaniel W
• Format: Journal Article
• Language: English
• Published: Springer 01.05.2016
• Subjects: Chemical properties; Identification and classification; Isotope analysis; Liquid chromatography; Mass spectrometry; Methods; Thioesters
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-016-9448-5

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 [.sup.13C.sub.3.sup.15N.sub.1] label derived from pantothenate into the CoA moiety or via variable [.sup.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-CoA thioesters by stable isotope dilution using the [.sup.13C.sub.3.sup.15N.sub.1] label and measurement of the incorporation of labeled carbon atoms derived from [.sup.13C.sub.6]-glucose, [.sup.13C.sub.5.sup.15N.sub.2]-glutamine, and [.sup.13C.sub.3]-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.