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.
- MeSH
- Brassica napus chemie MeSH
- brassinosteroidy analýza izolace a purifikace MeSH
- chromatografie afinitní metody MeSH
- imobilizační protilátky chemie MeSH
- imunosorbenty chemie MeSH
- regulátory růstu rostlin analýza izolace a purifikace MeSH
- rostlinné extrakty chemie MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
Brassinosteroids (BRs) are a class of steroid plant hormones that participate with other plant hormones in the regulation of numerous developmental processes, including root and shoot growth, vascular differentiation, fertility, and seed germination. A characteristic feature of all plant hormones, including BRs, is that their concentration is extremely low in plant tissues and, therefore, the methods dealing with their determination belong to ultra-trace analysis, for which very sensitive analytical tools are needed. The analysis of natural BRs is essential when their functions and roles in plant growth and development are to be elucidated. Here, we describe a reliable protocol for high-throughput extraction and purification of BRs. The procedure consists of two solid-phase extraction steps and provides selective enrichment and efficient cleanup of these compounds from complex plant extracts. The protocol is designed for sensitive liquid chromatography-tandem mass spectrometry-based method for simultaneous detection of 22 naturally occurring BRs, including their biosynthetic precursors and most of their biologically active metabolites, without need for derivatization.
- MeSH
- Arabidopsis chemie MeSH
- Brassica napus chemie MeSH
- brassinosteroidy chemie izolace a purifikace MeSH
- chromatografie kapalinová MeSH
- chromatografie plynová MeSH
- extrakce na pevné fázi metody MeSH
- hmotnostní spektrometrie MeSH
- regulátory růstu rostlin chemie izolace a purifikace MeSH
- rostlinné extrakty chemie MeSH
- Publikační typ
- časopisecké články MeSH
The triterpenoid plant hormones brassinosteroids (BRs) are believed to influence almost every aspect of plant growth and development. We have developed a sensitive mass spectrometry-based method for the simultaneous profiling of twenty-two naturally occurring brassinosteroids including biosynthetic precursors and the majority of biologically active metabolites. Using ultra-high performance liquid chromatographic (UHPLC) analysis, the run time was reduced up to three times (to 9 min) in comparison to standard HPLC BRs analyses, the retention time stability was improved to 0.1-0.2 % RSD and the injection accuracy was increased to 1.1-4.9 % RSD. The procedures for extraction and for two-step purification based on solid-phase extraction (SPE) were optimised in combination with subsequent UHPLC analysis coupled to electrospray ionisation tandem mass spectrometry (ESI-MS/MS) using Brassica flowers and Arabidopsis plant tissue extracts. In multiple reaction monitoring (MRM) mode, the average detection limit for BRs analysed was close to 7 pg, and the linear range covered up to 3 orders of magnitude. The low detection limits for this broad range of BR metabolites enabled as little as 50 mg of plant tissue to be used for quantitative analyses. The results of determinations exploiting internal standards showed that this approach provides a high level of practicality, reproducibility and recovery. The method we have established will enable researchers to gain a better understanding of the dynamics of the biosynthesis and metabolism of brassinosteroids and their modes of action in plant growth and development.
- MeSH
- Arabidopsis chemie MeSH
- Brassica napus chemie MeSH
- brassinosteroidy analýza MeSH
- extrakce na pevné fázi metody MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- limita detekce MeSH
- reprodukovatelnost výsledků MeSH
- rostlinné extrakty chemie MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
The Dothideomycete Leptosphaeria maculans, a worldwide fungal pathogen of oilseed rape (Brassica napus), secretes a broad spectrum of molecules into the cultivation medium during growth in vitro. Here, candidate elicitor molecules, which induce resistance in B. napus to L. maculans, were identified in the cultivation medium. The elicitation activity was indicated by increased transcription of pathogenesis-related gene 1 (PR1) and enhanced resistance of B. napus plants to the invasion of L. maculans. The elicitation activity was significantly lowered when the cultivation medium was heated to 80°C. Active components were further characterized by specific cleavage with the proteolytic enzymes trypsin and proteinase K and with glycosidases α-amylase and β-glucanase. The elicitor activity was eliminated by proteolytic digestion while glycosidases had no effect. The filtered medium was fractionated by either ion-exchange chromatography or isoelectric focusing. Mass spectrometry analysis of the most active fractions obtained by both separation procedures revealed predominantly enzymes that can be involved in the degradation of plant cell wall polysaccharides. This is the first study searching for L. maculans-specific secreted elicitors with a potential to be used as defense-activating agents in the protection of B. napus against L. maculans in agriculture. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:918-928, 2016.
- MeSH
- antifungální látky analýza metabolismus farmakologie MeSH
- Ascomycota účinky léků metabolismus MeSH
- Brassica napus chemie metabolismus mikrobiologie MeSH
- chromatografie iontoměničová MeSH
- fungální proteiny analýza metabolismus MeSH
- hmotnostní spektrometrie MeSH
- kultivační média chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Klíčová slova
- vepřové sádlo - nejstabilnější, řepkový olej, oxidační produkty, polymerační produkty,
- MeSH
- Brassica napus * chemie metabolismus škodlivé účinky MeSH
- chromatografie * metody využití MeSH
- Helianthus * chemie metabolismus škodlivé účinky MeSH
- kvalita jídla * MeSH
- lidé MeSH
- nenasycené mastné kyseliny * MeSH
- oleje rostlin * analýza chemie metabolismus škodlivé účinky MeSH
- oxidace-redukce * MeSH
- refraktometrie * metody normy využití MeSH
- slunečnicový olej MeSH
- statistika jako téma MeSH
- tokoferoly * analýza metabolismus MeSH
- triglyceridy * analýza chemie metabolismus škodlivé účinky MeSH
- tuky * analýza chemie metabolismus škodlivé účinky MeSH
- vysoká teplota * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
A robust, reliable and high-throughput method for extraction and purification of gibberellins (GAs), a group of tetracyclic diterpenoid carboxylic acids that include endogenous growth hormones, from plant material was developed. The procedure consists of two solid-phase extraction steps (Oasis(®) MCX-HLB and Oasis(®) MAX) and gives selective enrichment and efficient clean-up of these compounds from complex plant extracts. The method was tested with plant extracts of Brassica napus and Arabidopsis thaliana, from which total recovery of internal standards of about 72% was achieved. A rapid baseline chromatographic separation of 20 non-derivatised GAs by ultra performance liquid chromatography is also presented where a reversed-phase chromatographic column Acquity CSH(®) and a mobile phase consisting of methanol and aqueous 10mM-ammonium formate is used. This method enables sensitive and precise quantitation of GAs by MS/MS in multiple-reaction monitoring mode (MRM) by a standard isotope dilution method. Optimal conditions, including final flow rate, desolvation temperature, desolvation gas flow, capillary and cone voltage for effective ionisation in the electrospray ion source were found. All studied GAs were determined as free acids giving dominant quasi-molecular ions of [M-H](-) with limits of detection ranging between 0.08 and 10 fmol and linear ranges over four orders of magnitude. Taking advantage of highly effective chromatographic separation of 20 GAs and very sensitive mass spectrometric detection, the presented bioanalytical method serves as a useful tool for plant biologists studying the physiological roles of these hormones in plant development.
- MeSH
- Arabidopsis chemie MeSH
- Brassica napus chemie MeSH
- chromatografie kapalinová metody MeSH
- extrakce na pevné fázi MeSH
- gibereliny analýza MeSH
- květy chemie MeSH
- rostlinné extrakty analýza MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- výhonky rostlin chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- Brassica napus chemie MeSH
- kyseliny linolenové analýza MeSH
- lidé MeSH
- oleje rostlin MeSH
- Check Tag
- lidé MeSH
