硼亲和Tip的非共价构建及其在内源性油菜素甾醇分析中的应用
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- 英文论文题名：4-Phenylaminomethyl-Benzeneboronate Acid Modified Tip Extraction for Determination of Brassinosteroids in Plant Tissues by Stable Isotope Labeling-Liquid Chromatography-Mass Spectrometry
- 论文作者：余磊 ; 丁俊 ; 冯钰锜
- 英文论文作者：Lei Yu ; Jun Ding ; Yu-Qi Feng ; Department of Chemistry ; Wuhan University
- 年：2016
- 作者机构：武汉大学化学与分子科学学院;
- 论文关键词：油菜素甾醇 ; 原位衍生 ; 硼亲和Tip ; LC-MS/MS
- 英文论文关键词：brassinosteroids ; in situ derivatization ; boronate affinity tip ; LC-MS
- 会议召开时间：2016-07-01
- 会议录名称：中国化学会第30届学术年会摘要集-第二十三分会：复杂样品分离分析
- 语种：中文
- 分类号：Q946
- 学会代码：ZGHY
- 会议名称：中国化学会第30届学术年会-第二十三分会 ; 复杂样品分离分析
- 会议地点：中国辽宁大连
- 主办单位：中国化学会
- 学会名称：中国化学会
- 页数：1
- 文件大小：290k
- 原文格式：D
- 会议级别：全国
摘要
植物组织中内源性油菜素甾醇的分析方法对其分子作用机理研究具有重要意义。然而,植物基质的复杂性和油菜素甾醇在植物体内的低含量,导致油菜素甾醇的检测是一项具有挑战的工作。我们通过集成硼亲和原理、tip微萃取、化学标记技术和LC-MS技术,建立了一种油菜素内酯分析的新方法。首先将衍生化试剂4-苯基氨基甲基苯硼酸(4-PAMBA)通过离子交换和疏水作用修饰于tip头中的C8-SO_3H混合模式吸附剂上,构建了基于硼亲和作用原理的tip头微萃取装置;以此为萃取装置同时实现了对植物中内源性油菜素甾醇的选择性萃取和衍生,然后采用碱性溶液解吸油菜素内酯的衍生化产物,进行LC-MS分析。为了解决油菜素甾醇同位素内标获取困难的问题,采用4-PAMBA-d_5标记的油菜素甾醇标品作为内标提高LC-MS检测的准确度。该分析方法具有植物样品用量少、简单快速、通量高、灵敏高、高选择性好等特点,可对50 mg(鲜重)水稻样品中BRs准确定量。该方法还被进一步用于研究水稻在镉胁迫下内源性油菜素甾醇的含量变化。
Monitoring brassinosteroids(BRs) has been of major interest as they play a crucial role in a variety of physiological processes in plants.However,the determination of endogenous BRs in plant tissue is still a challenging task due to their low abundance and the complex matrix of plant tissues.In this study,a single step strategy by combining tip extraction and in situ derivatization was proposed for BR analysis.In the proposed strategy,a mixed mode sorbent(C8-SO_3H) in-tip was modified with 4-phenylaminomethyl-benzeneboronate acid(4-PAMBA) through cation exchange and hydrophobic interactions,and then used as a boronate affinity media to selectively capture and purify BRs from plant extract through the reaction of boronate acid groups of4-PAMBA and cis-diol on BRs.The BRs-4-PAMBA derivatives formed were easily eluted from the C8-SO_3H tip by nullifying the ion exchange and hydrophobic interactions using ammonia acetonitrile,followed by LC-MS/MS analysis.BR standards,isotopically labeled with d_5-4-phenylaminomethyl-benzeneboronate acid were introduced to improve the assay precision of LC-MS/MS.Under the optimized conditions,the overall process could be completed within 1hour,which is greatly improved in speed compared with previously reported protocols.In addition,the detection sensitivities of labeled BRs were improved by over 2000 folds compared with unlabeled BRs,thus the consumption of plant materials was reduced to 50 mg.Finally,the proposed method was applied for the investigation of BRs response in rice towards Cd stress.
Monitoring brassinosteroids(BRs) has been of major interest as they play a crucial role in a variety of physiological processes in plants.However,the determination of endogenous BRs in plant tissue is still a challenging task due to their low abundance and the complex matrix of plant tissues.In this study,a single step strategy by combining tip extraction and in situ derivatization was proposed for BR analysis.In the proposed strategy,a mixed mode sorbent(C8-SO_3H) in-tip was modified with 4-phenylaminomethyl-benzeneboronate acid(4-PAMBA) through cation exchange and hydrophobic interactions,and then used as a boronate affinity media to selectively capture and purify BRs from plant extract through the reaction of boronate acid groups of4-PAMBA and cis-diol on BRs.The BRs-4-PAMBA derivatives formed were easily eluted from the C8-SO_3H tip by nullifying the ion exchange and hydrophobic interactions using ammonia acetonitrile,followed by LC-MS/MS analysis.BR standards,isotopically labeled with d_5-4-phenylaminomethyl-benzeneboronate acid were introduced to improve the assay precision of LC-MS/MS.Under the optimized conditions,the overall process could be completed within 1hour,which is greatly improved in speed compared with previously reported protocols.In addition,the detection sensitivities of labeled BRs were improved by over 2000 folds compared with unlabeled BRs,thus the consumption of plant materials was reduced to 50 mg.Finally,the proposed method was applied for the investigation of BRs response in rice towards Cd stress.
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