Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China.
Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan 430072, P. R. China.
Anal Chem. 2021 Jul 20;93(28):9904-9911. doi: 10.1021/acs.analchem.1c02058. Epub 2021 Jul 6.
Epoxy/dihydroxy-oxylipins are important biologically active compounds that are mainly formed from polyunsaturated fatty acids (PUFAs) in the reactions catalyzed by the cytochrome P450 (CYP 450) enzyme. The analysis of epoxy/dihydroxy-oxylipins would be helpful to gain insights into their landscape in living organisms and provide a reference for the biological studies of these compounds. In this work, we employed chemical labeling-assisted liquid chromatography (LC) coupled with high-resolution mass spectrometry (CL-LC-HRMS) to establish a highly sensitive and specific method for screening and annotating epoxy/dihydroxy-oxylipins in biological samples. The isotope reagents 2-dimethylaminoethylamine (DMED) and DMED- were employed to label epoxy/dihydroxy-oxylipins containing carboxyl groups so as to improve the analysis selectivity and MS detection sensitivity of epoxy/dihydroxy-oxylipins. Based on a pair of diagnostic ions with a mass-to-charge ratio (/) difference of 15.995 originating from the fragmentation of derivatives via high-energy collision dissociation (HCD), the potential epoxy/dihydroxy-oxylipins were rapidly screened from the complex matrix. Furthermore, the epoxy/dihydroxy groups could be readily localized by the diagnostic ion pairs, which enabled us to accurately annotate the epoxy/dihydroxy-oxylipins detected in biological samples. The applicability of our method was demonstrated by profiling epoxy/dihydroxy-oxylipins in human serum and heart samples from mice with high-fat diet (HFD). By the proposed method, a total of 32 and 62 potential epoxy/dihydroxy-oxylipins including 42 unreported ones were detected from human serum and the mice heart sample, respectively. Moreover, the relative quantitative results showed that most of the potential epoxy/dihydroxy-oxylipins, especially the oxidation products of linoleic acid (LA) or α-linolenic acid (ALA), were significantly decreased in the heart of mice with HFD. Our developed method is of high specificity and sensitivity and thus is a promising tool for the identification of novel epoxy/dihydroxy-oxylipins in biological samples.
环氧化物/二羟基氧化脂类是一类重要的生物活性化合物，主要由多不饱和脂肪酸（PUFAs）在细胞色素 P450（CYP 450）酶催化的反应中形成。分析环氧化物/二羟基氧化脂类有助于深入了解它们在生物体内的分布情况，并为这些化合物的生物研究提供参考。在这项工作中，我们采用化学标记辅助液相色谱（LC）与高分辨质谱（CL-LC-HRMS）联用的方法，建立了一种高灵敏度和高特异性的方法，用于筛选和注释生物样品中的环氧化物/二羟基氧化脂类。我们使用 2-二甲氨基乙胺（DMED）和 DMED-对含有羧基的环氧化物/二羟基氧化脂类进行标记，以提高环氧化物/二羟基氧化脂类的分析选择性和 MS 检测灵敏度。基于通过高能碰撞解离（HCD）衍生化后产生的一对质荷比（m/z）差为 15.995 的特征离子对，我们可以从复杂基质中快速筛选出潜在的环氧化物/二羟基氧化脂类。此外，通过特征离子对可以很容易地确定环氧化物/二羟基氧化脂类的位置，从而可以准确注释在生物样品中检测到的环氧化物/二羟基氧化脂类。通过对高脂饮食（HFD）诱导的小鼠血清和心脏样本中环氧化物/二羟基氧化脂类的分析，验证了该方法的适用性。通过所提出的方法，从人血清和小鼠心脏样本中分别检测到了 32 种和 62 种潜在的环氧化物/二羟基氧化脂类，其中包括 42 种未报道过的物质。此外，相对定量结果表明，大多数潜在的环氧化物/二羟基氧化脂类，特别是亚油酸（LA）或α-亚麻酸（ALA）的氧化产物，在 HFD 诱导的小鼠心脏中显著减少。我们开发的方法具有高特异性和灵敏度，是一种很有前途的生物样品中环氧化物/二羟基氧化脂类鉴定工具。
