The potential of ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC/MS) and ultrahigh-performance supercritical fluid chromatography (UHPSFC) coupled to negative-ion electrospray ionization mass spectrometry (ESI-MS) for the analysis of 46 oxylipins and 2 fatty acid standards is compared in terms of their chromatographic resolution with the emphasis on distinguishing isobaric interferences and the method sensitivity. UHPLC provides the baseline separation of 24 isobaric oxylipins within 13min, while UHPSFC enables the separation of only 20 isobaric oxylipins within 8min. Moreover, the UHPLC/ESI-MS method provides an average improvement of sensitivity by 3.5-fold. A similar trend is observed in the analysis of human plasma samples, but lower ion suppression effects caused by lysophospholipids (LPL) are observed in case of UHPSFC/ESI-MS due to better separation of LPL. Both methods are fully applicable for the analysis of oxylipins, but UHPLC/ESI-MS method is preferred due to better separation and higher sensitivity, which results in the identification of 31 oxylipins in human plasma based on available standards and additional tentative 20 identifications based on accurate m/z values and the fragmentation behavior known from the literature.
- MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- lidé MeSH
- oxylipiny krev chemie MeSH
- rozpouštědla chemie MeSH
- superkritická fluidní chromatografie metody MeSH
- teplota MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Jasmonates (JAs) are plant hormones that integrate external stress stimuli with physiological responses. (+)-7-iso-JA-L-Ile is the natural JA ligand of COI1, a component of a known JA receptor. The upstream JA biosynthetic precursor cis-(+)-12-oxo-phytodienoic acid (cis-(+)-OPDA) has been reported to act independently of COI1 as an essential signal in several stress-induced and developmental processes. Wound-induced increases in the endogenous levels of JA/JA-Ile are accompanied by two to tenfold increases in the concentration of OPDA, but its means of perception and metabolism are unknown. To screen for putative OPDA metabolites, vegetative tissues of flowering Arabidopsis thaliana were extracted with 25% aqueous methanol (v/v), purified by single-step reversed-phase polymer-based solid-phase extraction, and analyzed by high throughput mass spectrometry. This enabled the detection and quantitation of a low abundant OPDA analog of the biologically active (+)-7-iso-JA-L-Ile in plant tissue samples. Levels of the newly identified compound and the related phytohormones JA, JA-Ile and cis-(+)-OPDA were monitored in wounded leaves of flowering Arabidopsis lines (Col-0 and Ws) and compared to the levels observed in Arabidopsis mutants deficient in the biosynthesis of JA (dde2-2, opr3) and JA-Ile (jar1). The observed cis-(+)-OPDA-Ile levels varied widely, raising questions concerning its role in Arabidopsis stress responses.
- MeSH
- Arabidopsis chemie MeSH
- cyklopentany chemie izolace a purifikace MeSH
- diazoniové sloučeniny MeSH
- isoleucin analogy a deriváty chemie MeSH
- květy chemie MeSH
- listy rostlin metabolismus MeSH
- nenasycené mastné kyseliny chemie izolace a purifikace metabolismus MeSH
- oxylipiny chemie izolace a purifikace MeSH
- pyridiny MeSH
- regulátory růstu rostlin metabolismus MeSH
- stereoizomerie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Stress-induced changes in phytohormone metabolite profiles have rapid effects on plant metabolic activity and growth. The jasmonates (JAs) are a group of fatty acid-derived stress response regulators with roles in numerous developmental processes. To elucidate their dual regulatory effects, which overlap with those of other important defence-signalling plant hormones such as salicylic acid (SA), abscisic acid (ABA) and indole-3-acetic acid (IAA), we have developed a highly efficient single-step clean-up procedure for their enrichment from complex plant matrices that enables their sensitive quantitative analysis using hyphenated mass spectrometry technique. The rapid extraction of minute quantities of plant material (less than 20mg fresh weight, FW) into cold 10% methanol followed by one-step reversed-phase polymer-based solid phase extraction significantly reduced matrix effects and increased the recovery of labile JA analytes. This extraction and purification protocol was paired with a highly sensitive and validated ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method and used to simultaneously profile sixteen stress-induced phytohormones in minute plant material samples, including endogenous JA, several of its biosynthetic precursors and derivatives, as well as SA, ABA and IAA.
- MeSH
- chromatografie kapalinová MeSH
- cyklopentany analýza chemie MeSH
- extrakce na pevné fázi MeSH
- fyziologický stres MeSH
- kyselina abscisová analýza chemie MeSH
- kyselina salicylová analýza chemie MeSH
- kyseliny indoloctové analýza MeSH
- listy rostlin chemie MeSH
- mastné kyseliny analýza chemie MeSH
- molekulární struktura MeSH
- oxylipiny analýza chemie MeSH
- regulátory růstu rostlin chemie izolace a purifikace fyziologie MeSH
- tandemová hmotnostní spektrometrie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lipoxygenázy (LOX, linoleate: oxygen oxidoreductases, EC 1.13.11.12) tvoria rodinu dioxygenáz, ktoré obsahujú nehémové, nesulfidové železo. Vyskytujú sa nielen u živočíchov, ale aj u rastlín. Ich prítomnosť bola dokázaná aj v koraloch, machu, hubách a v niektorých baktériách. LOX katalyzujú polohovo- a stereo- špecifickú inzerciu molekulového kyslíka do molekuly nenasýtenej mastnej kyseliny s cis,cis-1,4-pentadiénovým systémom za vzniku príslušných hydroperoxidových derivátov. Tento krok dioxygenácie vyúsťuje do kaskády reakcií, ktoré sa označujú ako lipoxygenázová (oktadekánová) cesta. Koncové produkty tejto cesty (nazývané oxylipíny) zohrávajú u rastlín významnú úlohu ako signálne molekuly pri hojení rán a pri obranných procesoch. V živočíšnych organizmoch sú zase zapojené do procesov zápalových reakcií, astmy a ochorení srdca.
Lipoxygenases (LOX, linoleate: oxygen oxidoreductases, EC 1.13.11.12) constitute a family of dioxygenases, which contain non-heme, non-sulfide iron. These enzymes occur not only in animals, but in plants as well. They have been detected in coral, moss, fungi and also in some bacteria. LOXs catalyse the regiospecific and stereospecific insertion of molecular oxygen into the molecule of polyunsaturated fatty acid with the cis,cis- -1,4-pentadiene system to yield the corresponding hydroperoxides. This step of dioxygenation leads to a cascade of reactions called the lipoxygenase (octadecanoid) pathway. The products of this pathway (called oxylipins) play an important role as signal molecules in wound healing and defence processes in plants. In animals they are involved in inflammation, asthma and heart diseases.
- MeSH
- biochemické jevy * MeSH
- buněčná diferenciace MeSH
- iniciace genetické transkripce MeSH
- lipoxygenasy * fyziologie MeSH
- membránové potenciály MeSH
- membránové transportní proteiny MeSH
- nenasycené mastné kyseliny chemie MeSH
- oxylipiny * chemie MeSH
- rostliny MeSH
- signální transdukce MeSH
- transkripční faktory MeSH
- Publikační typ
- přehledy MeSH
