Skip to Content
Merck
  • Stable isotope dilution liquid chromatography/mass spectrometry analysis of cellular and tissue medium- and long-chain acyl-coenzyme A thioesters.

Stable isotope dilution liquid chromatography/mass spectrometry analysis of cellular and tissue medium- and long-chain acyl-coenzyme A thioesters.

Rapid communications in mass spectrometry : RCM (2015-01-07)
Nathaniel W Snyder, Sankha S Basu, Zinan Zhou, Andrew J Worth, Ian A Blair
ABSTRACT

Acyl-Coenzyme A (CoA) thioesters are the principal form of activated carboxylates in cells and tissues. They are employed as acyl carriers that facilitate the transfer of acyl groups to lipids and proteins. Quantification of medium- and long-chain acyl-CoAs represents a significant bioanalytical challenge because of their instability. Stable isotope dilution liquid chromatography/selected reaction monitoring-mass spectrometry (LC/SRM-MS) provides the most specific and sensitive method for the analysis of CoA species. However, relevant heavy isotope standards are not available and they are challenging to prepare by chemical synthesis. Stable isotope labeling by essential nutrients in cell culture (SILEC), developed originally for the preparation of stable isotope labeled short-chain acyl-CoA thioester standards, has now been extended to medium-chain and long-chain acyl-CoAs and used for LC/SRM-MS analyses. Customized SILEC standards with >98% isotopic purity were prepared using mouse Hepa 1c1c7 cells cultured in pantothenic-free media fortified with [(13) C3 (15) N1 ]-pantothenic acid and selected fatty acids. A SILEC standard in combination with LC/SRM-MS was employed to quantify cellular concentrations of arachidonoyl-CoA (a representative long-chain acyl-CoA) in two human colon cancer cell lines. A panel of SILEC standards was also employed in combination LC/SRM-MS to quantify medium- and long-chain acyl-CoAs in mouse liver. This new SILEC-based method in combination with LC/SRM-MS will make it possible to rigorously quantify medium- and long-chain acyl-CoAs in cells and tissues. The method will facilitate studies of medium- and long-chain acyl-CoA dehydrogenase deficiencies as well as studies on the role of medium- and long-chain acyl-CoAs in cellular metabolism.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Acetic acid-12C2, 99.9 atom % 12C
USP
Calcium Pantothenate, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
Methanol, anhydrous, 99.8%
Sigma-Aldrich
Acetonitrile solution, contains 0.1 % (v/v) trifluoroacetic acid, suitable for HPLC
Supelco
Acetic acid, analytical standard
Sigma-Aldrich
D-Pantothenic acid hemicalcium salt, ≥98.0%
Supelco
Methanol, analytical standard
Supelco
Ammonium formate, eluent additive for LC-MS, LiChropur, ≥99.0%
Sigma-Aldrich
Ammonium formate, BioUltra, ≥99.0% (calc. based on dry substance, NT)
Sigma-Aldrich
Ammonium formate, ≥99.995% trace metals basis
Sigma-Aldrich
Acetic acid, ≥99.5%, FCC, FG
Sigma-Aldrich
Calcium pantothenate, meets USP testing specifications, monograph mol wt. 476.53 (C18H32CaN2O10)
Sigma-Aldrich
D-Pantothenic acid hemicalcium salt, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture
Calcium pantothenate, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
Acetic acid, natural, ≥99.5%, FG
Sigma-Aldrich
Acetic acid solution, suitable for HPLC
Sigma-Aldrich
Methanol, BioReagent, ≥99.93%
Sigma-Aldrich
Methanol, NMR reference standard
Sigma-Aldrich
Isopropyl alcohol, ≥99.7%, FCC, FG
Sigma-Aldrich
Acetic acid, for luminescence, BioUltra, ≥99.5% (GC)
Supelco
Residual Solvent - Acetonitrile, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Acetic acid, glacial, ACS reagent, ≥99.7%
Sigma-Aldrich
Methanol, ACS reagent, ≥99.8%
Sigma-Aldrich
Methanol, Absolute - Acetone free
Sigma-Aldrich
Methanol, ACS spectrophotometric grade, ≥99.9%
Sigma-Aldrich
Ammonium formate, reagent grade, 97%
Supelco
Methanol, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Acetic acid, glacial, puriss., 99-100%
Sigma-Aldrich
Acetic acid, glacial, puriss., meets analytical specification of Ph. Eur., BP, USP, FCC, 99.8-100.5%
Sigma-Aldrich
Acetic acid, glacial, ReagentPlus®, ≥99%