Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, 430072, Hubei, China.
Anal Bioanal Chem. 2019 Mar;411(8):1623-1632. doi: 10.1007/s00216-019-01612-9. Epub 2019 Feb 4.
Brassinosteroids (BRs) are endogenous plant growth-promoting hormones affecting growth and development during the entire life cycle of plants. Naturally occurring BRs can be classified into C-, C-, or C-BRs based on the nature of the alkyl groups occupying the C-24 position in the side chain of the 5a-cholestane carbon skeleton. However, while C-BRs exhibit similar bioactivities to C- and C-BRs, the biosynthetic pathways of C-BRs in plants have not yet been clearly characterized. In addition to a lack of biochemical and enzymatic evidence regarding the biosynthetic pathways of C-BRs, even most of the intermediate compounds on their pathways have not been explored and identified due to the lower endogenous levels of C-BRs. Therefore, the development of highly sensitive analytical methods is essential for studying the biosynthetic pathways and physiological functions of C-BRs. Accordingly, this study establishes qualitative and quantitative methods for identifying and detecting C-, C-, and C-BRs using a newly synthesized boronic acid reagent denoted as 2-methyl-4-phenylaminomethylphenylboronic acid (2-methyl-4-PAMBA) in conjunction with liquid chromatography-mass spectrometry (LC-MS). Labeling with 2-methyl-4-PAMBA provides derivatives with excellent stability, and the detection sensitivities of BRs, particularly for C-BRs, are dramatically improved. The limits of detection (with a signal-to-noise ratio of 3) for six BRs, including 2 C-BRs (28-norCS and 28-norBL), 3 C-BRs (CS, BL, and TY), and a single C-BR (28-homoBL), are found to be 0.10-1.68 pg/mL after labeling with 2-methyl-4-PAMBA. Finally, the proposed analytical method is successfully applied for the detection of endogenous BRs in small mass samples of Oryza sativa seedlings, Rape flowers, Arabidopsis shoots, and Arabidopsis flowers. In addition, a method for profiling potential BRs in plants is also developed using LC-MS in multiple reaction monitoring scan mode assisted by 2-methyl-4-PAMBA and 2-methyl-4-PAMBA-d labeling. The developed method is able to identify 10 potential BRs in a Rape flower extract. The proposed quantitative and qualitative methods established by 2-methyl-4-PAMBA labeling are helpful for facilitating an understanding of the physiological functions and biosynthetic pathways of BRs, particularly for C-BRs. Graphical abstract.
油菜素甾醇（BRs）是一种内源性植物生长促进激素，影响植物整个生命周期的生长和发育。根据侧链 5a-胆甾烷碳骨架中 C-24 位占据的烷基基团的性质，天然存在的 BRs 可分为 C-、C-或 C-BRs。然而，虽然 C-BRs 表现出与 C-和 C-BRs 相似的生物活性，但植物中 C-BRs 的生物合成途径尚未得到明确表征。除了缺乏关于 C-BRs 生物合成途径的生化和酶学证据外，由于 C-BRs 的内源性水平较低，甚至其途径上的大多数中间化合物也尚未被探索和鉴定。因此，开发高灵敏度的分析方法对于研究 C-BRs 的生物合成途径和生理功能至关重要。因此，本研究使用一种新合成的硼酸试剂，即 2-甲基-4-苯氨基甲基苯硼酸（2-甲基-4-PAMBA），结合液相色谱-质谱（LC-MS），建立了定性和定量鉴定和检测 C-、C-和 C-BRs 的方法。用 2-甲基-4-PAMBA 标记提供了具有优异稳定性的衍生物，并且 BRs 的检测灵敏度，特别是对 C-BRs 的检测灵敏度得到了显著提高。用 2-甲基-4-PAMBA 标记后，六种 BRs（包括 2 种 C-BRs（28-降 CS 和 28-降 BL）、3 种 C-BRs（CS、BL 和 TY）和 1 种 C-BR（28-高同 BL））的检测限（信噪比为 3）为 0.10-1.68 pg/mL。最后，该分析方法成功应用于检测水稻幼苗、油菜花、拟南芥芽和拟南芥花中小质量样本中的内源性 BRs。此外，还开发了一种使用 LC-MS 在 2-甲基-4-PAMBA 和 2-甲基-4-PAMBA-d 标记辅助的多重反应监测扫描模式下对植物中潜在 BRs 进行分析的方法。该方法能够鉴定油菜花提取物中的 10 种潜在 BRs。2-甲基-4-PAMBA 标记建立的定量和定性方法有助于促进对 BRs，特别是 C-BRs 的生理功能和生物合成途径的理解。
