title: 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.
authors.affiliation: AJ Drexel Autism Institute, Drexel University, 3020 Market Street Suite 560, Philadelphia, PA, 19104, USA.
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Frey
authors.affiliation: AJ Drexel Autism Institute, Drexel University, 3020 Market Street Suite 560, Philadelphia, PA, 19104, USA.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Feldman
authors.affiliation: AJ Drexel Autism Institute, Drexel University, 3020 Market Street Suite 560, Philadelphia, PA, 19104, USA.
authors.forename: Sophie
authors.initials: S
authors.lastname: Trefely
authors.affiliation: Bristol-Myers Squibb Pharmaceutical Candidate Optimization, Wallingford, CT, 06492, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Worth
authors.affiliation: Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA, 02115, USA.
authors.forename: Sankha S
authors.initials: SS
authors.lastname: Basu
authors.affiliation: AJ Drexel Autism Institute, Drexel University, 3020 Market Street Suite 560, Philadelphia, PA, 19104, USA. nws28@drexel.edu.
authors.forename: Nathaniel W
authors.initials: NW
authors.lastname: Snyder
mesh_terms: D000214:Acyl Coenzyme A / Q000032:analysis*; D002247:Carbon Isotopes / Q000737:chemistry*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D004952:Esters / Q000737:chemistry*; D006801:Humans; D007553:Isotope Labeling
chemical_list: D000214:Acyl Coenzyme A; D002247:Carbon Isotopes; D004952:Esters
keywords: Bioanalytical methods; Coenzyme A; Mass spectrometry; Metabolism; Stable isotope labeling