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.
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DOI：
10.1002/rcm.6958
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发表时间：
2014-08-30
影响因子：
2
通讯作者：
Blair, Ian A.
中科院分区：
文献类型：
--
作者：
Snyder, Nathaniel W.;Basu, Sankha S.;Zhou, Zinan;Worth, Andrew J.;Blair, Ian A.
酰基辅酶A（CoA）硫酯是细胞和组织中活化羧酸盐的主要形式。它们被用作酰基载体，促进酰基转移到脂质和蛋白质。由于中链和长链酰基辅酶A的不稳定性，它们的定量是一个重大的生物分析挑战。稳定同位素稀释-液相色谱-选择反应监测-质谱（LC-SRM/MS）为CoA种类的分析提供了最特异、最灵敏的方法。然而，相关的重同位素标准品不可用，并且通过化学合成制备它们具有挑战性。最初用于制备稳定同位素标记的短链酰基辅酶A硫酯标准品的细胞培养物中必需营养素稳定同位素标记（SILEC）现已扩展至中链和长链酰基辅酶A，并用于LC-SRM/MS分析。使用在用[13 C315 N1]-泛酸和选定脂肪酸强化的无泛酸培养基中培养的小鼠Hepa 1c 1c 7细胞制备同位素纯度> 98%的定制SILEC标准品。采用SILEC标准品与LC-SRM/MS组合来定量两种人结肠癌细胞系中花生四烯酸酰-CoA（代表性长链酰基-CoA）的细胞浓度。一组SILEC标准品也用于LC-SRM/MS组合中，以定量小鼠肝脏中的中链和长链酰基辅酶A。这种新的基于SILEC的方法与LC-SRM/MS相结合，将使严格定量细胞和组织中的中链和长链酰基辅酶A成为可能。该方法将促进中长链酰基辅酶A脱氢酶缺乏的研究以及中长链酰基辅酶A在细胞代谢中的作用的研究。
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-chain 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 [13C315N1]-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.
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