A General Method for Quantification and Discovery of Acyl Groups Attached to Acyl Carrier Proteins in Fatty Acid Metabolism Using LC-MS/MS
A General Method for Quantification and Discovery of Acyl Groups Attached to Acyl Carrier Proteins in Fatty Acid Metabolism Using LC-MS/MS
复制标题
使用 LC-MS/MS 定量和发现脂肪酸代谢中酰基载体蛋白上附着的酰基的通用方法
DOI：
10.1105/tpc.19.00954
复制
发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Allen, Doug K.
中科院分区：
文献类型：
--
作者：
Nam, Jeong-Won;Jenkins, Lauren M.;Li, Jia;Evans, Bradley S.;Jaworski, Jan G.;Allen, Doug K.
酰基载体蛋白（ACP）是生命系统中脂肪酸生物合成的支架，使其对全面了解脂质代谢至关重要。然而，不存在评估单个酰基-ACP的准确定量方法。我们开发了一个强大的方法来量化酰基-ACP的皮克级。我们成功地鉴定了酰基-ACP延伸中间体（3-羟基酰基-ACP和2，3-反式-烯酰基-ACP）和意想不到的中链（C10：1，C14：1）和多不饱和长链（C16：3）酰基-ACP，表明该方法的灵敏度以及目前对脂质代谢和ACP功能的描述是如何不完整的。这样的ACP可能是重要的燃料中链脂质生产和突出了解不多的脂质重塑事件在叶绿体。该方法广泛适用于在植物和细菌中发现的II型脂肪酸合酶系统以及来自哺乳动物和真菌的线粒体，因为它利用了ACP中高度保守的Asp-Ser-Leu-Asp氨基酸序列，酰基基团附着于该序列。我们的方法允许使用液相色谱-串联质谱法与de novo生成的标准品和同位素稀释策略进行灵敏的定量，并将填补我们理解中的空白，通过定量探索脂肪酸生物合成过程提供见解，以获得最佳生物燃料，可再生原料以及健康和疾病的医学研究。
Acyl carrier proteins (ACPs) are the scaffolds for fatty acid biosynthesis in living systems, rendering them essential to a comprehensive understanding of lipid metabolism. However, accurate quantitative methods to assess individual acyl-ACPs do not exist. We developed a robust method to quantify acyl-ACPs to the picogram level. We successfully identified acyl-ACP elongation intermediates (3-hydroxyacyl-ACPs and 2,3-trans-enoyl-ACPs) and unexpected medium-chain (C10:1, C14:1) and polyunsaturated long-chain (C16:3) acyl-ACPs, indicating both the sensitivity of the method and how current descriptions of lipid metabolism and ACP function are incomplete. Such ACPs are likely important to medium-chain lipid production for fuels and highlight poorly understood lipid remodeling events in the chloroplast. The approach is broadly applicable to type II fatty acid synthase systems found in plants and bacteria as well as mitochondria from mammals and fungi because it capitalizes on a highly conserved Asp-Ser-Leu-Asp amino acid sequence in ACPs to which acyl groups attach. Our method allows for sensitive quantification using liquid chromatography–tandem mass spectrometry with de novo–generated standards and an isotopic dilution strategy and will fill a gap in our understanding, providing insights through quantitative exploration of fatty acid biosynthesis processes for optimal biofuels, renewable feedstocks, and medical studies in health and disease.