Fatty acids (FAs) possess highly diverse structures and can be divided into saturated and unsaturated classes. For unsaturated FAs, both the numbers and positions of carbon-carbon double bond (C=C) determine their biological functions. Abnormal levels of FA isomers have been reported to be involved in various disease development, such as cancer. Despite numerous advances in lipidomics, simultaneous quantifying and pinpointing C=C bond positions in a high-throughput manner remains a challenge. Here we conducted epoxidation of C=C bonds of unsaturated FAs followed by the conjugation with isobaric SUGAR tags. With the assistance of LC-MS, FA isomers with the same masses were separated on the C18 column and individually subjected to MS/MS fragmentation. Upon higher-energy collisional dissociation, not only reporter ions for multiplexed quantification but also diagnostic ions for C=C localization were generated at the same time, allowing quantitative analyses of different unsaturated FA isomers in samples. The performance of this approach including epoxidation, labeling efficiencies, quantitation accuracy, and capability to pinpoint C=C bond position were evaluated. To evaluate our method, free FA extracts from healthy human serum were used to demonstrate the feasibility of this method for complex sample analysis. Finally, this method was also applied to investigate the changes of unsaturated FA isomers between heathy human and Alzheimer's disease (AD) patient serum.

脂肪酸 (FAs) 具有高度多样化的结构，可分为饱和和不饱和两类。对于不饱和脂肪酸，碳碳双键（C=C）的数量和位置决定了它们的生物学功能。据报道，FA 异构体的异常水平与各种疾病的发展有关，例如癌症。尽管脂质组学取得了许多进展，但以高通量方式同时量化和精确定位 C=C 键位置仍然是一个挑战。在这里，我们对不饱和 FA 的 C=C 键进行环氧化，然后与同量异位 SUGAR 标签进行共轭。在 LC-MS 的帮助下，具有相同质量的 FA 异构体在 C18 柱上被分离，并分别进行 MS/MS 碎裂。在高能碰撞解离时，不仅可以同时生成用于多重定量的报告离子，还可以生成用于 C=C 定位的诊断离子，从而可以对样品中不同的不饱和 FA 异构体进行定量分析。评估了这种方法的性能，包括环氧化、标记效率、定量准确度和精确定位 C=C 键位置的能力。为了评估我们的方法，使用来自健康人血清的游离 FA 提取物来证明该方法在复杂样品分析中的可行性。最后，该方法还应用于研究健康人与阿尔茨海默病（AD）患者血清之间不饱和FA异构体的变化。评估了这种方法的性能，包括环氧化、标记效率、定量准确度和精确定位 C=C 键位置的能力。为了评估我们的方法，使用来自健康人血清的游离 FA 提取物来证明该方法在复杂样品分析中的可行性。最后，该方法还应用于研究健康人与阿尔茨海默病（AD）患者血清之间不饱和FA异构体的变化。评估了这种方法的性能，包括环氧化、标记效率、定量准确度和精确定位 C=C 键位置的能力。为了评估我们的方法，使用来自健康人血清的游离 FA 提取物来证明该方法在复杂样品分析中的可行性。最后，该方法还应用于研究健康人与阿尔茨海默病（AD）患者血清之间不饱和FA异构体的变化。