SILEC: a protocol for generating and using isotopically labeled coenzyme A mass spectrometry standards.
SILEC: a protocol for generating and using isotopically labeled coenzyme A mass spectrometry standards.
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DOI：
10.1038/nprot.2011.421
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发表时间：
2011-12-08
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影响因子：
14.8
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细胞培养中必需营养素的稳定同位素标记（SILEC）最近被开发用于产生同位素标记的辅酶A（CoA）和短链酰基-CoA硫酯。这是通过修改广泛使用的技术稳定同位素标记的氨基酸在细胞培养中，包括[13 C315 N]-泛酸（维生素B5），辅酶A的前体，而不是同位素标记的氨基酸。缺乏从头泛酸合成途径允许测量的CoA种类的有效和接近完全的标记。该方案提供了在哺乳动物和昆虫细胞中生成稳定的同位素标记的短链酰基辅酶A内标物的分步方法，以及如何在基于稳定同位素稀释质谱的分析中使用它们的说明。故障排除指南，以及未标记和标记的CoA种类的列表也包括在内。该方案代表了从标记的必需营养素如泛酸盐产生稳定同位素内标物的原型。SILEC标准的生成和使用大约需要2-3周。
Stable isotope labeling by essential nutrients in cell culture (SILEC) was recently developed to generate isotopically labeled coenzyme A (CoA) and short-chain acyl-CoA thioesters. This was accomplished by modifying the widely used technique of stable isotope labeling by amino acids in cell culture to include [13C315N]-pantothenate (vitamin B5), a CoA precursor, instead of the isotopically labeled amino acids. The lack of a de novo pantothenate synthesis pathway allowed for efficient and near-complete labeling of the measured CoA species. This protocol provides a step-by-step approach for generating stable isotope-labeled short-chain acyl-CoA internal standards in mammalian and insect cells as well as instructions on how to use them in stable isotope dilution mass spectrometric-based analyses. Troubleshooting guidelines, as well as a list of unlabeled and labeled CoA species, are also included. This protocol represents a prototype for generating stable isotope internal standards from labeled essential nutrients such as pantothenate. The generation and use of SILEC standards takes approximately 2–3 weeks.