Analytical approach for characterization of cadmium-induced thiol peptides—a case study using Chlamydomonas reinhardtii

Abstract

Phytochelatins (PC) were described earlier to play a role in metal detoxification in Chlamydomonas reinhardtii but were not clearly identified. The focus of this case study was to identify PC synthesized by C. reinhardtii exposed to Cd. Only low intracellular concentrations of cadmium (85 nmol g(-1) fresh weight) were sufficient to cause significant changes in thiol peptide pools. Thus, results showed a progressive decline of the glutathione content, accompanied by an induction of phytochelatins. Not only canonic phytochelatins but for the first time also the iso-phytochelatins CysPC(n) and PC(2)Ala were identified in this unicellular green alga using electrospray ionization quadrupole time-of-flight tandem mass spectrometry. Additionally, CysPC(n)desGly, PC(n)desGly, CysPC(n)Glu, and PC(2)Glu were found throughout MS analysis. Also, low abundant PCs could be detected due to the high sample preconcentration combined with little sample amounts (0.3 microL min(-1)) necessary for electrospray. Identified PCs had a maximum number of 5 gamma-glutamyl cysteine (gamma-GluCys) units. Thiol peptides of higher molecular masses suggesting PC(n) with n > 5 could be identified as intermolecular oxidation products of smaller PCs. Thiols may easily be oxidized. Therefore, PCs were reduced prior to MS analysis. Dithiothreitol and tris(2-carboxyethyl) phosphine were compared concerning their reduction effort.

Arabidopsis thaliana phytochelatin synthase 2 is constitutively active in vivo and can rescue the growth defect of the PCS1-deficient cad1-3 mutant on Cd-contaminated soil

“…In order to evaluate if and how AtPCS2 contributes to the PC pool and thereby potentially to the buffering of free metal ions within the cell, it was necessary to establish a highly sensitive method for the detection of trace amounts of PCs. Coupling of reversed-phase LC to ESI-QTOF-MS enabled such PC detection (Bräutigam et al , 2009; Simmons et al , 2009; Tennstedt et al , 2009). In this study, mBrB-derivatized thiols, reduced with TCEP, were analysed via UPLC-ESI-QTOF-MS.…”

Characterization of the Phytochelatin Synthase of Schistosoma mansoni

“…at a flow rate of 1 ml/min. Elutes were collected and assayed with 500 µM 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) in 50 mM potassium phosphate (pH 8) buffer to detect the free sulfhydryls of cysteine, γ-GluCys, GSH and PCs at 412 nm in Multiskan Spectrum plate reader [34], [35], [36]. Synthetic PC2 (γ-GluCys-γ-GluCysGly), PC3 (γ-GluCys-γ-GluCys-γ-GluCysGly) and PC4 (γ-GluCys-γ-GluCys-γ-GluCys-γ-GluCysGly were prepared at the proteomics core facility at the University of Illinois-Chicago.…”

Physiological characterization of cadmium‐exposed Chlamydomonas reinhardtii

“…Recently, we could demonstrate that the alga also produces isoforms of PCs, mainly CysPC 2 (C(EC) 2 G) and CysPC 3 (C(EC) 3 G) (Bräutigam et al . 2009, 2010b).…”

“…Also, the green alga Stigeoclonium tenue synthesizes these PC isoforms in response to zinc (Pawlik‐Skowronska 2003). Recently, we hypothesized a CysPCs generation via cleavage of the γ ‐peptide bond of PCs (Bräutigam et al . 2009), which is reasonable as GSH and glutathione S‐conjugates can be degraded to CysGly or Cys‐S‐conjugates, respectively (Martin & Slovin 2000).…”