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Production of stable isotope-labeled acyl-coenzyme A thioesters by yeast stable isotope labeling by essential nutrients in cell culture

机译：通过细胞培养中必需营养素通过酵母稳定同位素标记生产稳定同位素标记的酰基辅酶A硫酯

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Acyl-coenzyme A (CoA) thioesters are key metabolites in numerous anabolic and catabolic pathways, including fatty acid biosynthesis and β-oxidation, the Krebs cycle, and cholesterol and isoprenoid biosynthesis. Stable isotope dilution-based methodology is the gold standard for quantitative analyses by mass spectrometry. However, chemical synthesis of families of stable isotope labeled metabolites such as acyl-coenzyme A thioesters is impractical. Previously, we biosynthetically generated a library of stable isotope internal standard analogs of acyl-CoA thioesters by exploiting the essential requirement in mammals and insects for pantothenic acid (vitamin B5) as a metabolic precursor for the CoA backbone. By replacing pantothenic acid in the cell media with commercially available [13C3 15N1]-pantothenic acid, mammalian cells exclusively incorporated [13C3 15N1]-pantothenate into the biosynthesis of acyl-CoA and acyl-CoA thioesters. We have now developed a much more efficient method for generating stable isotope labeled CoA and acyl-CoAs from [13C3 15N1]-pantothenate using Stable Isotope Labeling by Essential nutrients in Cell culture (SILEC) in Pan6 deficient yeast cells. Efficiency and consistency of labeling were also increased, likely due to the stringently defined and reproducible conditions used for yeast culture. The yeast SILEC method greatly enhances the ease of use and accessibility of labeled CoA thioesters and also provides proof-of-concept for generating other labeled metabolites in yeast mutants.
机译：酰基辅酶A（CoA）硫酯是许多合成代谢和分解代谢途径中的关键代谢产物，包括脂肪酸生物合成和β-氧化，克雷布斯循环以及胆固醇和类异戊二烯生物合成。基于同位素的稳定稀释方法是质谱定量分析的金标准。然而，化学合成稳定的同位素标记的代谢物家族，例如酰基辅酶A硫酯，是不切实际的。以前，我们通过利用哺乳动物和昆虫中对于泛酸（维生素B5）作为CoA主链代谢前体的基本要求，生物合成了酰基辅酶A硫酯的稳定同位素内标类似物库。通过用市售的[ 13 C3 15 N1]-泛酸替换细胞培养基中的泛酸，哺乳动物细胞专门掺入[ 13 C3 < sup> 15 N1]-泛酸酯用于酰基辅酶A和酰基辅酶A硫酯的生物合成。现在，我们已经开发了一种效率更高的方法，该方法使用稳定的同位素标记法从[ 13 C3 15 N1]-泛酸中生成稳定的同位素标记的CoA和酰基辅酶A Pan6缺陷酵母细胞中的细胞培养（SILEC）。标记的效率和一致性也提高了，这可能是由于酵母培养所用的条件严格且可重复。酵母SILEC方法大大提高了标记CoA硫酯标记的易用性和可及性，还为在酵母突变体中产生其他标记代谢物提供了概念验证。

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