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

A method based on pneumatically assisted electrospray ionisation tandem mas s 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)(2)Gly (PC2), (GluCys)(3)Gly (PC3), and (GluCys)( 4)Gly (PC4) standards prepared from glutathione by enzymatically (gamma-glu tamylcysteine dipeptyl transpeptidase) assisted biosynthesis in the presenc e of Cd2+. Phytochelatins were found to ionize readily to produce a charact eristic mono-protonated ion. The collision-induced dissociation (CID) of th is ion followed by mass spectrometry (MS-MS mode) allowed the determination of the amino acid sequence of each of the PCs. Calibration curves were lin ear up to a concentration of 2 mu g ml(-1) in the MS and MS-MS modes with t he detection limits at the low ng ml(-1) level. The method was applied to t he determination of phytochelatin peptides biosynthesized by a number of pl ant cell cultures exposed to the Cd stress. The results agreed with those o btained 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.