Fatty acids (FAs) are essential cellular components and play important roles in various biological processes. Importantly, FAs produced by microorganisms from renewable sugars are considered sustainable substrates for biodiesels and oleochemicals. Their complex structures and diverse functional roles in biochemical processes necessitate the development of efficient and accurate methods for their quantitative analysis. Here, we developed a novel method for relative quantification of FAs by combining 12-plex isobaric -methyl cine-derivatized thyleediamine (DiLeuEN) labeling and microchip capillary electrophoresis-mass spectrometry (CE-MS). This method enables simultaneous quantification of 12 samples in a single MS analysis. DiLeuEN labeling introduced tertiary amine center structure into FAs, which makes them compatible with the positive mode separation of commercial microchip CE systems and further improves the sensitivity. The CE separation parameters were optimized, and the quantification accuracy was assessed using FA standards. Microchip CE-MS detection exhibited high sensitivity with a femtomole level detection limit and a total analysis time within 8 min. Finally, the applicability of our method to complex biological samples was demonstrated by analyzing FAs produced by four industrially relevant yeast strains (, , and ). The analysis time for each sample is less than 1 min. This work addresses the current challenges in the field by introducing a method that combines microchip-based capillary electrophoresis separation with multiplex isobaric labeling. Our method not only offers remarkable sensitivity and rapid analysis speed but also the capability to quantify fatty acids across multiple samples simultaneously, which holds significant potential for extensive application in FA quantitative studies in diverse research areas, promising an enhanced understanding of FA functions and mechanisms.

中文翻译：

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通过 12 重同量异位标记和微芯片毛细管电泳对脂肪酸进行高通量相对定量 - 质谱法


脂肪酸（FA）是必需的细胞成分，在各种生物过程中发挥着重要作用。重要的是，微生物从可再生糖中产生的 FA 被认为是生物柴油和油脂化学品的可持续底物。它们的复杂结构和在生化过程中的不同功能作用需要开发高效、准确的方法来进行定量分析。在这里，我们通过结合 12 重同量异位甲基电影衍生甲状腺二胺 (DiLeuEN) 标记和微芯片毛细管电泳-质谱 (CE-MS) 开发了一种新的 FA 相对定量方法。该方法可在单次 MS 分析中同时定量 12 个样品。 DiLeuEN标记将叔胺中心结构引入FA中，使其与商用微芯片CE系统的正模式分离兼容，并进一步提高了灵敏度。优化了 CE 分离参数，并使用 FA 标准评估了定量准确性。 Microchip CE-MS 检测表现出高灵敏度，检测限达到飞摩尔水平，总分析时间在 8 分钟内。最后，通过分析四种工业相关酵母菌株（、、和）产生的 FA，证明了我们的方法对复杂生物样品的适用性。每个样品的分析时间不到 1 分钟。这项工作通过引入一种将基于微芯片的毛细管电泳分离与多重同量异位标记相结合的方法来解决该领域当前的挑战。 我们的方法不仅具有卓越的灵敏度和快速的分析速度，而且能够同时定量多个样品中的脂肪酸，这在不同研究领域的 FA 定量研究中具有广泛应用的巨大潜力，有望增强对 FA 功能和机制的理解。