Profiling of potential brassinosteroids in different tissues of rape flower by stable isotope labeling - liquid...

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.