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.
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.
Vacchina
Véronique
V
Group of Bio-Inorganic Analytical Chemistry, UMR 5034 CNRS, Helioparc, 2, Av. Pr. Angot, F-64053 Pau, France.
Mari
Stéphane
S
Czernic
Pierre
P
Marquès
Laurence
L
Pianelli
Katia
K
Schaumlöffel
Dirk
D
Lebrun
Michel
M
Lobiński
Ryszard
R
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D015723:Gene Library; D005816:Genetic Complementation Test; D013058:Mass Spectrometry / Q000379:methods; D009532:Nickel / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D031227:Thlaspi / Q000235:genetics / Q000378:metabolism*
D018076:DNA, Complementary; D009532:Nickel