Stable isotope labeling by essential nutrients in cell culture for preparation of labeled coenzyme A and its thioesters

Abstract

Stable isotope dilution mass spectrometry (MS) represents the gold standard for quantification of endogenously formed cellular metabolites. Although coenzyme A (CoA) and acyl-CoA thioester derivatives are central players in numerous metabolic pathways, the lack of a commercially available isotopically labeled CoA limits the development of rigorous MS-based methods. In this study, we adapted stable isotope labeling by amino acids in cell culture (SILAC) methodology to biosynthetically generate stable isotope labeled CoA and thioester analogues for use as internal standards in liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) assays. This was accomplished by incubating murine hepatocytes (Hepa 1c1c7) in media in which pantothenate (a precursor of CoA) was replaced with [(13)C(3)(15)N(1)]-pantothenate. Efficient incorporation into various CoA species was optimized to >99% [(13)C(3)(15)N(1)]-pantothenate after three passages of the murine cells in culture. Charcoal-dextran-stripped fetal bovine serum (FBS) was found to be more efficient for serum supplementation than dialyzed or undialyzed FBS, due to lower contaminating unlabeled pantothenate content. Stable isotope labeled CoA species were extracted and utilized as internal standards for CoA thioester analysis in cell culture models. This methodology of stable isotope labeling by essential nutrients in cell culture (SILEC) can serve as a paradigm for using vitamins and other essential nutrients to generate stable isotope standards that cannot be readily synthesized.

Figure 1

Metabolic pathway for CoA biosynthesis from pantothenate (vitamin B5). Enzymes: (A) pantothenate kinase (rate-limiting); (B) phosphopantothenoyl cysteine synthetase; (C) phosphopantothenoyl cysteine decarboxylase; (D) phosphopantetheine adenylyltransferase; (E) dephospho-CoA kinase. [¹³C] and [¹⁵N] atoms are marked in red.

Figure 2

LC/constant neutral loss MS analysis of hepatocytes extracts with a neutral loss of m/z 507 showing CoA (MH⁺, m/z 768), acetyl-CoA (MH⁺, m/z 810), propionyl-CoA (MH⁺, m/z 824), succinyl-CoA (MH⁺, m/z 868), and HMG-CoA (MH⁺, m/z 912).

Figure 3

Analysis of short-chain CoA thioesters. LC/MRM-MS chromatograms of (A) CoA thioester standards (1 pmol each) and (B) acidic extracts from 1 plate of Hepa 1c1c7 cells.

Figure 4

Stable isotope labeling of CoA thioesters in Hepa 1c1c7 cells grown in [¹³C₃¹⁵N₁]-pantothenate (1 mg/L). (A) LC/constant neutral loss MS analysis with a neutral loss of m/z 507 from hepatocytes extracts after 24 h of pantothenate labeling. (B) Time course of pantothenate incorporation into CoASH using LC/MRM-MS analysis.

Figure 5

Preparation of a specific stable isotope labeled CoA thioester stable isotope standard: CoA extracts from stable isotope labeled cells (A) untreated and (B) treated with 10 mM propionate for 60 min.

Figure 6

Changes in intracellular CoA thioester levels in Hepa 1c1c7 after propionate treatment: untreated (black) and 10 mM propionate for 1 h (gray). CoA thioesters extracted from control and treated labeled cells were combined and used as stable isotope internal standards in measuring short-chain acyl-CoA species. ∗∗ p < 0.005, ∗∗∗ p < 0.00005.