Speciation of Nickel in a Hyperaccumulating Plant by High-Performance Liquid Chromatography−Inductively Coupled Plasma Mass Spectrometry and Electrospray MS/MS Assisted by Cloning Using Yeast Complementation

• Published in: Analytical chemistry (Washington) 2003-06, Vol.75 (11), p.2740-2745
• Main Authors: Vacchina, Véronique, Mari, Stéphane, Czernic, Pierre, Marquès, Laurence, Pianelli, Katia, Schaumlöffel, Dirk, Lebrun, Michel, Łobiński, Ryszard
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Biological and medical sciences; Chemical Sciences; Chromatography, High Pressure Liquid - methods; Cloning, Molecular; Diverse techniques; DNA, Complementary - analysis; Fundamental and applied biological sciences. Psychology; Gene Library; Genetic Complementation Test; Mass Spectrometry - methods; Molecular and cellular biology; Nickel - analysis; Nickel - chemistry; Nickel - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Spectrometry, Mass, Electrospray Ionization; Thlaspi - genetics; Thlaspi - metabolism
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac020704m

A novel analytical approach based on a combination of multidimensional hyphenated techniques and cloning of the Ni-resistance gene using yeast complementation screens was developed for the identification of nickel species in a Thlaspi caerulescens hyperaccumulating plant. The presence of an unknown strong Ni complex was demonstrated by size exclusion HPLC−capillary electrophoresis with ICPMS detection. The Ni-containing peak was characterized by electrospray MS (m/z 360) and shown by collision-induced dissociation MS to be a chelate with a tricarboxylic amino acid ligand. To identify the species and demonstrate its functional character, a cDNA library was constructed from T. caerulescens, expressed in the yeast, and screened on a toxic Ni2+ medium. The extract from the surviving transformant culture gave identical HPLC−ICPMS, CZE−ICPMS, and ES MS/MS data and contained a cDNA insert homologous to the nicotianamine synthase gene. This observation allowed the identification of nicotianamine as the nickel-binding ligand. The presence of the Ni−nicotianamine complex was ultimately demonstrated by comparing tandem mass spectra of the plant and yeast extracts with those of a synthetic standard.