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

Frey, Alexander; Feldman, Daniel; Trefely, Sophie; Worth, Andrew; Basu, Sankha; Snyder, Nathaniel

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 [CN] label derived from pantothenate into the CoA moiety or via variable [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 [CN] label and measurement of the incorporation of labeled carbon atoms derived from [C]-glucose, [CN]-glutamine, and [C]-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. [Figure not available: see fulltext.]

LABORATORY techniques; CULTURE techniques (Biology); PHOTOSPECTROMETRY; DIALYSIS (Chemistry); OPTICAL measurements

Analytical & Bioanalytical Chemistry, 2016, Vol 408, Issue 13, p3651

1618-2642

Academic Journal

10.1007/s00216-016-9448-5