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 C₂₇-, C₂₈-, or C₂₉-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 C₂₇-BRs exhibit similar bioactivities to C₂₈- and C₂₉-BRs, the biosynthetic pathways of C₂₇-BRs in plants have not yet been clearly characterized. In addition to a lack of biochemical and enzymatic evidence regarding the biosynthetic pathways of C₂₇-BRs, even most of the intermediate compounds on their pathways have not been explored and identified due to the lower endogenous levels of C₂₇-BRs. Therefore, the development of highly sensitive analytical methods is essential for studying the biosynthetic pathways and physiological functions of C₂₇-BRs. Accordingly, this study establishes qualitative and quantitative methods for identifying and detecting C₂₇-, C₂₈-, and C₂₉-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 C₂₇-BRs, are dramatically improved. The limits of detection (with a signal-to-noise ratio of 3) for six BRs, including 2 C₂₇-BRs (28-norCS and 28-norBL), 3 C₂₈-BRs (CS, BL, and TY), and a single C₂₉-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-d₅ 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 C₂₇-BRs.

Open image in new window

油菜素类固醇（BRs）是内源性植物生长激素，会影响植物整个生命周期的生长和发育。基于在5a-胆甾烷碳骨架的侧链中占据C-24位的烷基的性质，天然存在的BR可分为C _27-，C _28-或C ₂₉ -BR。然而，尽管C ₂₇ -BR表现出与C _28-和C ₂₉ -BR相似的生物活性，但尚未明确表征植物中C ₂₇ -BR的生物合成途径。除了缺乏有关C ₂₇生物合成途径的生化和酶促证据外-BRs，甚至由于其内源性C ₂₇ -BRs水平较低，甚至尚未探索和鉴定其路径上的大多数中间化合物。因此，开发高灵敏度的分析方法对于研究C ₂₇ -BR的生物合成途径和生理功能至关重要。因此，本研究建立了定性和定量的方法来鉴定和检测C _27-，C _28-和C ₂₉-BRs使用新合成的称为2-甲基-4-苯基氨基甲基苯基硼酸（2-甲基-4-PAMBA）的硼酸试剂与液相色谱-质谱（LC-MS）结合使用。用2-甲基-4-PAMBA进行标记可提供具有出色稳定性的衍生物，并且显着提高了BR的检测灵敏度，尤其是对C ₂₇ -BR的灵敏度。六个BR的检出限（信噪比为3），包括2个C ₂₇ -BR（28-norCS和28-norBL），3个C ₂₈ -BR（CS，BL和TY），用2-甲基-4-PAMBA标记后，发现单个C ₂₉ -BR（28-homoBL）为0.10–1.68 pg / mL。最后，所提出的分析方法已成功地用于检测小质量样品中的内源性BRs。水稻稻苗，油菜花，拟南芥芽和拟南芥花。此外，还使用LC-MS在2-甲基-4-PAMBA和2-甲基-4-PAMBA- d ₅标记辅助下的多反应监测扫描模式下开发了一种用于分析植物中潜在BR的方法。所开发的方法能够鉴定出强奸花提取物中的10种潜在BR 。通过2-甲基-4-PAMBA标记建立的拟议的定量和定性方法有助于促进对BRs的生理功能和生物合成途径的理解，特别是对于C ₂₇ -BRs。

在新窗口中打开图像