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      Immunoaffinity chromatography combined with tandem mass spectrometry: A new tool for the selective capture and analysis of brassinosteroid plant hormones.

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          Abstract

          Brassinosteroids (BRs) are plant-specific steroid hormones that play essential roles in the regulation of many important physiological processes in plant life. Their extremely low concentrations (~pmoles/g FW) in plant tissue and huge differences in polarity of individual members within the BR family hamper their detection and quantification. To address this problem, an immunoaffinity sorbent with broad specificity and high capacity for different BR metabolites containing a monoclonal antibody (mAb) against a BR spacer (20S)-2α,3α-dihydroxy-7-oxa-7α-homo-5α-pregnane-6-one-20 carboxylic acid (BR4812) was used for the rapid and highly selective isolation of endogenous BRs containing a 2α,3α-diol in ring A from minute plant samples. This enrichment procedure was successfully applied as a sample preparation method prior to quantitative analysis of BRs in real plant tissues by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Use of immunoaffinity chromatography (IAC) increased the sensitivity of the UHPLC-MS/MS analysis owing to improvements in the BR signal-to-noise ratio (S/N) and matrix factor (MF). Although MF values of BRs analyzed in classical samples ranged from 8.9% to 47.4%, MF values for the IAC purified samples reached 44.5-96.6%. Thus, the developed IAC-UHPLC-MS/MS approach was shown to be a simple, robust, effective and extremely fast procedure requiring minute amounts of plant samples suitable for the quantitative profiling of many BR metabolites, helping to overcome the major problems associated with their determination in very complex plant matrices.

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          Author and article information

          Journal
          Talanta
          Talanta
          Elsevier BV
          1873-3573
          0039-9140
          Aug 01 2017
          : 170
          Affiliations
          [1 ] Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, The Czech Academy of Sciences & Faculty of Science, Palacký University, Šlechtitelů 27, CZ-783 71 Olomouc, Czech Republic. Electronic address: jana.oklestkova@upol.cz.
          [2 ] Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, The Czech Academy of Sciences & Faculty of Science, Palacký University, Šlechtitelů 27, CZ-783 71 Olomouc, Czech Republic. Electronic address: tarkowska@ueb.cas.cz.
          [3 ] Veterinary Research Institute, Hudcova 70, CZ-621 00 Brno, Czech Republic. Electronic address: eyer@vri.cz.
          [4 ] Institute of Organic Chemistry and Biochemistry, The Czech Academy of Sciences, Flemingovo nám. 2, CZ-166 10 Prague 6, Czech Republic. Electronic address: elbert@uochb.cas.cz.
          [5 ] Institute of Organic Chemistry and Biochemistry, The Czech Academy of Sciences, Flemingovo nám. 2, CZ-166 10 Prague 6, Czech Republic. Electronic address: ales.marek@uochb.cas.cz.
          [6 ] Veterinary Research Institute, Hudcova 70, CZ-621 00 Brno, Czech Republic. Electronic address: smrzova@vri.cz.
          [7 ] Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, The Czech Academy of Sciences & Faculty of Science, Palacký University, Šlechtitelů 27, CZ-783 71 Olomouc, Czech Republic. Electronic address: ondrej.novak@upol.cz.
          [8 ] Veterinary Research Institute, Hudcova 70, CZ-621 00 Brno, Czech Republic. Electronic address: franek@vri.cz.
          [9 ] Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Kuprevich Str., 5/2, 220141 Minsk, Belarus. Electronic address: vz@iboch.bas-net.by.
          [10 ] Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Institute of Experimental Botany, The Czech Academy of Sciences & Faculty of Science, Palacký University, Šlechtitelů 27, CZ-783 71 Olomouc, Czech Republic. Electronic address: miroslav.strnad@upol.cz.
          Article
          S0039-9140(17)30469-1
          10.1016/j.talanta.2017.04.044
          28501193
          8614b6a5-84c5-4f41-acc9-8208d67dfd0f
          History

          Brassica napus,Brassinosteroids,Enzyme immunoassay,Immunoaffinity chromatography,Liquid chromatography-tandem mass spectrometry,Monoclonal antibodies

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