Haslam Richard P, Larson Tony R
Department of Plant Science, Rothamsted Research, Harpenden, UK.
Department of Biology, University of York, York, UK.
Methods Mol Biol. 2021;2295:203-218. doi: 10.1007/978-1-0716-1362-7_12.
The acyl-CoA pool is pivotal in cellular metabolism. The ability to provide reliable estimates of acyl-CoA abundance and distribution between molecular species in plant tissues and microalgae is essential to our understanding of lipid metabolism and acyl exchange. Acyl-CoAs are typically found in low abundance and require specific methods for extraction, separation and detection. Here we describe methods for acyl-CoA extraction and measurement in plant tissues and microalgae, with a focus on liquid chromatography hyphenated to detection techniques including ultraviolet (UV), fluorescence and mass spectrometry (MS). We address the resolution of isobaric species and the selection of columns needed to achieve this, including the analysis of branched chain acyl-CoA thioesters. For MS analyses, we describe diagnostic ions for the identification of acyl-CoA species and how these can be used for both discovery of new species (data dependent acquisition) and routine quantitation (triple quadrupole MS with multiple reaction monitoring).
酰基辅酶A库在细胞代谢中起着关键作用。能够可靠地估计植物组织和微藻中酰基辅酶A的丰度以及不同分子种类之间的分布，对于我们理解脂质代谢和酰基交换至关重要。酰基辅酶A通常含量较低，需要特定的提取、分离和检测方法。在此，我们描述了植物组织和微藻中酰基辅酶A的提取和测量方法，重点是与检测技术联用的液相色谱法，这些检测技术包括紫外（UV）、荧光和质谱（MS）。我们探讨了等压物质的分离以及实现这一目的所需的色谱柱选择，包括支链酰基辅酶A硫酯的分析。对于质谱分析，我们描述了用于鉴定酰基辅酶A种类的诊断离子，以及如何将这些离子用于新种类的发现（数据依赖型采集）和常规定量分析（具有多反应监测功能的三重四极杆质谱）。
