Quantification of Acyl-Acyl Carrier Proteins for Fatty Acid Synthesis Using LC-MS/MS
Quantification of Acyl-Acyl Carrier Proteins for Fatty Acid Synthesis Using LC-MS/MS
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DOI：
10.1007/978-1-0716-1362-7_13
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发表时间：
2021-01-01
期刊：
影响因子：
--
通讯作者：
Allen, Doug K.
中科院分区：
文献类型：
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作者：
Jenkins, Lauren M.;Nam, Jeong-Won;Allen, Doug K.
脂肪酸生物合成循环基于酰基载体蛋白（ACP）支架，其中通过一系列重复的酶促步骤在链延长过程中添加两个碳乙酰基。通过用硫酯酶水解或通过酰基转移酶将酰基直接转移至甘油磷脂来终止链延伸。用于分析连接到ACP的酰基链的浓度的方法是缺乏的，但将为脂质代谢的研究提供信息。我们描述了一种方法来配置文件和量化的酰基-ACP在植物，细菌和线粒体的动物和真菌，代表II型脂肪酸生物合成系统的水平。II型系统的ACP在携带酰基链的4 '-磷酸泛酰巯基乙胺基臂的连接位点处具有高度保守的Asp-Ser-Leu-Asp（DSLD）氨基酸序列。保守序列的三个氨基酸可以使用乙酰基蛋白酶从蛋白质的其余部分切割下来。因此，部分纯化的蛋白质可以被酶促水解以产生经由4 '-磷酸泛酰巯基乙胺基连接到三肽的酰基链。在电离和碎裂之后，通过使用多反应监测方法的三重四极杆质谱仪检测相应的碎片离子。将大量产生的N-15同位素标记的酰基-ACP与同位素稀释策略一起使用，以定量连接至酰基载体蛋白支架的每个酰基的绝对水平。
The fatty acid biosynthetic cycle is predicated on an acyl carrier protein (ACP) scaffold where two carbon acetyl groups are added in a chain elongation process through a series of repeated enzymatic steps. The chain extension is terminated by hydrolysis with a thioesterase or direct transfer of the acyl group to a glycerophospholipid by an acyltransferase. Methods for analysis of the concentrations of acyl chains attached to ACPs are lacking but would be informative for studies in lipid metabolism. We describe a method to profile and quantify the levels of acyl-ACPs in plants, bacteria and mitochondria of animals and fungi that represent Type II fatty acid biosynthetic systems. ACPs of Type II systems have a highly conserved Asp-Ser-Leu-Asp (DSLD) amino acid sequence at the attachment site for 4'-phosphopantetheinyl arm carrying the acyl chain. Three amino acids of the conserved sequence can be cleaved away from the remainder of the protein using an aspartyl protease. Thus, partially purified protein can be enzymatically hydrolyzed to produce an acyl chain linked to a tripeptide via the 4'-phosphopantetheinyl group. After ionization and fragmentation, the corresponding fragment ion is detected by a triple quadrupole mass spectrometer using a multiple reaction monitoring method. N-15 isotopically labeled acyl-ACPs generated in high amounts are used with an isotope dilution strategy to quantify the absolute levels of each acyl group attached to the acyl carrier protein scaffold.