Characterisation and determination of phytochelatins in plant extracts by electrospray tandem mass spectrometry

Abstract

A method based on pneumatically assisted electrospray ionisation tandem mass spectrometry (ESI MS-MS) was developed for the identification, sequencing and determination of phytochelatin (PC) peptides in plant tissue and plant cell cytosols. The ionization and fragmentation conditions were optimized using a series of (GluCys)₂Gly (PC₂), (GluCys)₃Gly (PC₃), and (GluCys)₄Gly (PC₄) standards prepared from glutathione by enzymatically (γ-glutamylcysteine dipeptyl transpeptidase) assisted biosynthesis in the presence of Cd²⁺. Phytochelatins were found to ionize readily to produce a characteristic mono-protonated ion. The collision-induced dissociation (CID) of this ion followed by mass spectrometry (MS-MS mode) allowed the determination of the amino acid sequence of each of the PCs. Calibration curves were linear up to a concentration of 2 µg ml^–1 in the MS and MS-MS modes with the detection limits at the low ng ml^–1 level. The method was applied to the determination of phytochelatin peptides biosynthesized by a number of plant cell cultures exposed to the Cd stress. The results agreed with those obtained by an independent procedure based on reversed-phase HPLC with post-column derivatization of the –SH groups with 5,5′-dithiobis-2-nitrobenzoic acid and spectrophotometric detection.