Characterization and quantification of thiol-peptides in Arabidopsis thaliana using combined dilution and high sensitivity HPLC-ESI-MS-MS

Publication date: August 2019Source: Phytochemistry, Volume 164Author(s): Erika Bellini, Marco Borsò, Camilla Betti, Laura Bruno, Andrea Andreucci, Monica Ruffini Castiglione, Alessandro Saba, Luigi Sanità di ToppiAbstractAlthough thiol-peptide compounds, such as reduced glutathione (GSH), γ-glutamylcysteine (γ-EC), and phytochelatins, play fundamental roles in plants, their analytical determination and characterization is still somewhat problematic, mainly due to their high polarity and oxidation propensity. Thus, in this work a reliable and sensitive HPLC-ESI-MS-MS method was developed, in order to simultaneously assay, within 14-min instrumental runs, γ-EC, GSH, and phytochelatins up to phytochelatin 4. This analytical method was validated in shoot and root extracts of the model plant Arabidopsis thaliana (Brassicaceae) and guaranteed accurate quantification by using specific isotope labelled-internal standards for both GSH and phytochelatins, as well as standards for external calibration. Good linearities in the method performance were observed (R > 0.99), with a dynamic range over three orders of magnitude in thiol-peptide concentrations. In MRM mode, the detection sensitivity of the thiol-peptides was equal to approximately 16, 6, 7, 13, 10 fmol for γ-EC, GSH, phytochelatin 2, phytochelatin 3, and phytochelatin 4, respectively (20 μl injection each). The reproducibility of the method was confirmed by high intra- and inter-day accuracy and precision values...
Source: Phytochemistry - Category: Chemistry Source Type: research