Brassinosteroids (BRs) play crucial roles in a variety of physiological processes in plants. The full elucidation of the functions of RBs relies on sensitive detection and accurate measurement of BRs in plants. However, the identification and quantification of BRs are challenging due to their low abundance as well as poor ionization efficiencies during mass spectrometry-based analysis. Herein, we developed a highly sensitive and selective strategy for profiling potential BRs in plants by stable isotope labeling liquid chromatography multiple reaction monitoring scan mass spectrometry (SIL-LC-MRM-MS) analysis. In the strategy, we used a pair of stable isotope labeling reagents 4-phenylaminomethyl-benzeneboronic acid (4-PAMBA) and d5-4-phenylaminomethyl-benzeneboronic acid (4-PAMBA-d5) that can react with C22-C23 cis-diol on BRs for profiling potential BRs in plant tissues. The 4-PAMBA and 4-PAMBA-d5 labeled BRs could generate two characteristic neutral loss under collision induced dissociation (CID), respectively, which is used to establish the MRM-based detection and screening. The precursor ions of BRs labeled with 4-PAMBA and 4-PAMBA-d5 were set according to the reported structures of BRs, and the corresponding product ions were predicted by subtracting the lost neutral loss. In this respect, corresponding precursor ions and product ions in MRM transitions are formed. The peak pairs with a fixed mass difference, similar retention times and intensities were assigned as potential BRs. Using the developed SIL-LC-MRM-MS strategy, we successfully found 13 potential BR in different tissues of rape flower. Taken together, the SIL-LC-MRM-MS analytical strategy is promising for profiling potential BRs as well as other compounds that have the same functional moiety from complex biological samples.

中文翻译：

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通过稳定同位素标记-液相色谱/质谱分析对油菜花不同组织中潜在的油菜素类固醇进行分析

油菜素类固醇 (BRs) 在植物的各种生理过程中起着至关重要的作用。RBs 功能的全面阐明依赖于对植物中 BRs 的灵敏检测和准确测量。然而，在基于质谱的分析过程中，BRs 的丰度低以及电离效率低，因此对 BR 的鉴定和定量具有挑战性。在此，我们开发了一种通过稳定同位素标记液相色谱多反应监测扫描质谱 (SIL-LC-MRM-MS) 分析来分析植物中潜在 BR 的高灵敏度和选择性策略。在战略中，我们使用了一对稳定同位素标记试剂 4-苯基氨基甲基-苯硼酸 (4-PAMBA) 和 d5-4-苯基氨基甲基-苯硼酸 (4-PAMBA-d5)，它们可以与 BR 上的 C22-C23 顺式二醇反应进行分析植物组织中潜在的 BR。4-PAMBA 和 4-PAMBA-d5 标记的 BRs 可以分别在碰撞诱导解离 (CID) 下产生两种特征中性丢失，用于建立基于 MRM 的检测和筛选。根据报道的 BRs 结构设置 4-PAMBA 和 4-PAMBA-d5 标记的 BRs 的前体离子，并通过减去失去的中性损失来预测相应的产物离子。在这方面，在 MRM 跃迁中形成了相应的母离子和产物离子。具有固定质量差的峰配对，相似的保留时间和强度被指定为潜在的 BR。使用开发的 SIL-LC-MRM-MS 策略，我们成功地在油菜花的不同组织中发现了 13 种潜在的 BR。总之，SIL-LC-MRM-MS 分析策略有望用于分析潜在的 BR 以及复杂生物样品中具有相同功能部分的其他化合物。