Stable isotope labeling by essential nutrients in cell culture for preparation of labeled coenzyme A and its thioesters.
Stable isotope labeling by essential nutrients in cell culture for preparation of labeled coenzyme A and its thioesters.
复制标题
通过细胞培养物中的必需营养素进行稳定同位素标记，用于制备标记的辅酶 A 及其硫酯。
DOI：
10.1021/ac1027353
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发表时间：
2011-02-15
影响因子：
7.4
通讯作者：
Blair, Ian A.
中科院分区：
文献类型：
--
作者：
Basu, Sankha S.;Mesaros, Clementina;Gelhaus, Stacy L.;Blair, Ian A.
稳定同位素稀释质谱 (MS) 代表了内源形成的细胞代谢物定量的金标准。尽管辅酶 A (CoA) 和酰基辅酶 A 硫酯衍生物是众多代谢途径的核心参与者，但缺乏市售同位素标记的辅酶 A 限制了严格的基于 MS 的方法的开发。在本研究中，我们采用细胞培养中氨基酸稳定同位素标记 (SILAC) 方法来生物合成生成稳定同位素标记的 CoA 和硫酯类似物，用作液相色谱/多反应监测质谱 (LC/MRM-MS) 测定中的内标。这是通过在用 [13C315N1]-泛酸盐取代泛酸盐（CoA 的前体）的培养基中孵育小鼠肝细胞 (Hepa 1c1c7) 来实现的。小鼠细胞培养三代后，有效掺入各种 CoA 物种的效率优化为 >99% [13C315N1]-泛酸。研究发现，木炭-葡聚糖剥离的胎牛血清（FBS）比透析或未透析的胎牛血清（FBS）更有效，因为其污染性未标记泛酸含量较低。提取稳定同位素标记的 CoA 物质并用作细胞培养模型中 CoA 硫酯分析的内标。这种通过细胞培养中必需营养素进行稳定同位素标记的方法 (SILEC) 可以作为使用维生素和其他必需营养素生成不易合成的稳定同位素标准品的范例。
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 [13C315N1]-pantothenate. Efficient incorporation into various CoA species was optimized to >99% [13C315N1]-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.
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DOI：
10.1016/j.bbalip.2009.09.024
发表时间：
2010-03
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Li LO;Klett EL;Coleman RA
通讯作者：
Coleman RA
影响因子：
14.8
作者：
Bennett, Bryson D.;Yuan, Jie;Rabinowitz, Joshua D.
通讯作者：
Rabinowitz, Joshua D.
影响因子：
2
作者：
Mesaros, Clementina;Lee, Seon Hwa;Blair, Ian A.
通讯作者：
Blair, Ian A.
DOI：
10.1016/j.jchromb.2004.05.012
发表时间：
2004-09-05
影响因子：
3
作者：
Mauriala, T;Herzig, KH;Auriola, S
通讯作者：
Auriola, S
影响因子：
1.6
作者：
Hayashi, Osamu;Satoh, Kiyoshi
通讯作者：
Satoh, Kiyoshi