Application Of Chemical Labeling Combined With Liquid Chromatography-Mass Spectrometry In Detection Of Signal Molecules In Plant

Signaling molecules in plants which transmit information between cells and within cells are essential for plant growth and development,allowing cells and tissues to sense and respond to stress in the external environment.Up to now,the signaling pathway are not clear.Therefore,quantification of signaling molecules is of great significance for further investigating the molecular interaction mechanism.In this paper,sensitive and accurate methods were developed to analysis of two kinds of signaling molecules including brassinosteroids(BRs)and sugar phosphates.Liquid chromatography-mass spectrometry(LC-MS)is an important tool for analysis of BRs and sugar phosphates because of its high selectivity and sensitivity.However,the difficulties still exist.Due to its extremely low content in plant(several pg/g to ng/g fresh weight),poor mass spectrometry response and plant matrix interference,the challenge in BRs analysis is to improve the sensitivity.Various pretreatment methods have been developed to enrich and label BRs.But there are still some deficiencies.First,the pretreatment step was fussy which should be further simplified.Then,the selectivity and sensitivity should be further improved.For sugar phosphates,due to their high polarity,improvement in separation on chromatography should be done as well as sensitivity in MS analysis.Up to now,several labeling reagents have been developed for analysis of sugar phosphates in animal cells and good results have been obtained.However,the analysis of disaccharide phosphates,such as trehalose-6-phosphate and sucrose-6-phosphate,need further exploration.Based on above problems,new methods which combined chemical labeling and LC-MS were developed to analysis of BRs and sugar phosphates in plant tissues.The specific contents are as follows:(1)Determination of BRs in plant tissues by solid phase boronate affinity labeling coupled with liquid chromatography-tandem mass spectrometry.MCX sorbent was first modified with the labeling reagent 2-(4-boronovenzyl)isoquinolin-2-ium(BBII)for selectively labeling and capturing of BRs at the same time through boronate affinity interaction.Afterwards,desorption and salt-induced phase transition extraction(SPTE)was carried out.In brief,90%acetone aqueous solution was served as elution solvent.As soon as 20-50 mg CH₃COONH₄ added,auto phase separation occurred because of salting-out effect.After being vigorously stirred and centrifuged,labeled BRs were desorbed from MCX and enriched in organic phase.Meanwhile,excessive labeling reagent was stayed in aqueous phase.This step ensured the labeled BRs to be desorbed and separated from the excess labeling reagent.Finally,the labeled BRs was collected and injected to LC-MS for analysis.In this study,no complicated material synthesis was required.The integration of labeling and extraction,desorption and SPTE made the method quick,sensitive and selective.Under the optimized conditions,the detection limit of the method was 1.4-2.8 pg/mL,and endogenous BRs were detected in small amount of plant tissues ranging from 2.8 mg to 20 mg fresh weight.(2)Determination of sugar phosphates in plant tissues by chemical labeling combined with hydrophilic interaction liquid chromatography-tandem mass spectrometry.8-(diazomethyl)quinoline(8-DMQ)was synthesized and utilized to label sugar phosphates including glucose-6-posphate,glucose-1-phosphate,trehalose-6-phosphate and sucrose-6-phosphate.After labeling,the chromatographic separation and sensitivity of the sugar phosphates were improved.The isomers could be well separated on a HILIC column,and the detection sensitivities of the sugar phosphates were improved by 3-fold to 114-fold with LODs ranging from 0.1 to 0.6 ng/mL.To our knowledge,it was the first time that chemical labeling applying in the analysis of sugar phosphates in plant tissues.Only 1 mg fresh weight of plant tissue was required for the analysis.