Concurrent profiling of indole-3-acetic acid, abscisic acid, and cytokinins and structurally related purines by high-performance-liquid-chromatography tandem electrospray mass spectrometry

Abstract

BackgroundCytokinins (CKs) are a group of plant growth regulators that are involved in several plant developmental processes. Despite the breadth of knowledge surrounding CKs and their diverse functions, much remains to be discovered about the full potential of CKs, including their relationship with the purine salvage pathway, and other phytohormones. The most widely used approach to query unknown facets of CK biology utilized functional genomics coupled with CK metabolite assays and screening of CK associated phenotypes. There are numerous different types of assays for determining CK quantity, however, none of these methods screen for the compendium of metabolites that are necessary for elucidating all roles, including purine salvage pathway enzymes in CK metabolism, and CK cross-talk with other phytohormones. Furthermore, all published analytical methods have drawbacks ranging from the required use of radiolabelled compounds, or hazardous derivatization reagents, poor sensitivity, lack of resolution between CK isomers and lengthy run times.ResultsIn this paper, a method is described for the concurrent extraction, purification and analysis of several CKs (freebases, ribosides, glucosides, nucleotides), purines (adenosine monophosphate, inosine, adenosine, and adenine), indole-3-acetic acid, and abscisic acid from hundred-milligram (mg) quantities of Arabidopsis thaliana leaf tissue. This method utilizes conventional Bieleski solvents extraction, solid phase purification, and is unique because of its diverse range of detectable analytes, and implementation of a conventional HPLC system with a fused core column that enables good sensitivity without the requirement of a UHPLC system. Using this method we were able to resolve CKs about twice as fast as our previous method. Similarly, analysis of adenosine, indole-3-acetic acid, and abscisic acid, was comparatively rapid. A further enhancement of the method was the utilization of a QTRAP 5500 mass analyzer, which improved upon several aspects of our previous analytical method carried out on a Quattro mass analyzer. Notable improvements included much superior sensitivity, and number of analytes detectable within a single run. Limits of detection ranged from 2 pM for (9G)Z to almost 750 pM for indole-3-acetic acid.ConclusionsThis method is well suited for functional genomics platforms tailored to understanding CK metabolism, CK interrelationships with purine recycling and associated hormonal cross-talk.

Phytohormone metabolism in human cells: Cytokinins are taken up and interconverted in HeLa cell culture

“…Quantification was achieved through isotope dilution analysis based on direct comparison of the analyte peak area to that of the recovered 2 H-labeled internal standards (Table 1). 34 The analysis of cis isomers of the zeatin-type CKs was evaluated relative to the recovery of labeled tZ-type CKs and the retention time of endogenous cZ-type CKs. Furthermore, the reported levels of 2MeSZ and 2MeSZR reflect a pool of cisand trans-methylthiolated (2MeS) zeatin.…”

“…Human cervical cancer cell pellet, supernatant, and medium blank samples were analyzed by high‐performance liquid chromatography‐positive electrospray ionization‐tandem mass spectrometry (HPLC‐(ESI+)‐MS/MS; Shimadzu LC‐10ADvp HPLC (Columbia, MD, USA) coupled with a QTrap 5500 mass spectrometer (Sciex Applied Biosystem, Concord, ON, Canada) with a turbo V‐spray ionization source. HPLC‐(ESI+)‐MS/MS methods and multiple reaction monitoring (MRM) channels, specific for each CK, were carried out as described by Farrow and Emery . A 20 µL sample aliquot was injected on a reversed‐phase C 18 column (Kinetex 2.6u C 18 100 A, 2.1 × 50mm ; Phenomenex, Torrance, CA), and CKs were eluted with an increasing gradient of solvent B (0.08% CH 3 CO 2 H in C 2 H 3 N) mixed with solvent A (0.08% CH 3 CO 2 H in H 2 O), at a flow rate of 0.4 mL/min.…”

“…The mass spectrometry data were processed using Analyst v. 1.6.2 software (AB Sciex, Concord, ON, Canada). Quantification was achieved through isotope dilution analysis based on direct comparison of the analyte peak area to that of the recovered 2 H‐labeled internal standards (Table ) . The analysis of cis isomers of the zeatin‐type CKs was evaluated relative to the recovery of labeled t Z‐type CKs and the retention time of endogenous c Z‐type CKs.…”

“…Canada) with a turbo V-spray ionization source. HPLC-(ESI+)-MS/MS methods and multiple reaction monitoring (MRM) channels, specific for each CK, were carried out as described by Farrow and Emery 34. A 20 µL sample aliquot was injected on a reversed-phase C 18 column (Kinetex 2.6u C 18 100 A, 2.1 × 50mm ; Phenomenex,…”

Euglena gracilis(Euglenophyceae) produces abscisic acid and cytokinins and responds to their exogenous application singly and in combination with other growth regulators

“…A modified protocol described by Quesnelle & Emery (2007) and Farrow & Emery (2012) was used. To prepare test cultures, 15 ml of Euglena stock culture was centrifuged (4000 rpm) for 5 min, the pellet was washed and re-suspended in 5 ml of DI water, and grown at 25°C and pH 7.5 for 48 hours.…”