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.

酰基载体蛋白（ACP）是生命系统中脂肪酸生物合成的支架，使它们对于全面了解脂质代谢至关重要。但是，不存在评估单个酰基ACP的准确定量方法。我们开发了一种鲁棒的方法来将酰基ACP定量到皮克级。我们成功地确定了酰基ACP延伸中间体（3-羟基酰基ACP和2,3-反式-enoyl-ACPs和意想不到的中链（C10：1，C14：1）和多不饱和长链（C16：3）酰基ACP，表明该方法的敏感性以及脂质代谢和ACP功能的最新描述不完整。此类ACP对燃料的中链脂质生产可能很重要，并突出了人们对叶绿体中脂质重塑事件了解不足。该方法广泛适用于植物和细菌以及哺乳动物和真菌的线粒体中发现的II型脂肪酸合酶系统，因为它利用了酰基上附着的ACP中高度保守的Asp-Ser-Leu-Asp氨基酸序列。我们的方法允许使用液相色谱-串联质谱和从头生成的标准液以及同位素稀释策略进行灵敏的定量分析，这将填补我们的空白，