Vacchina Véronique, Mari Stéphane, Czernic Pierre, Marquès Laurence, Pianelli Katia, Schaumlöffel Dirk, Lebrun Michel, Lobiński Ryszard
Group of Bio-Inorganic Analytical Chemistry, UMR 5034 CNRS, Helioparc, 2, Av. Pr. Angot, F-64053 Pau, France.
Anal Chem. 2003 Jun 1;75(11):2740-5. 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.
开发了一种基于多维联用技术与利用酵母互补筛选克隆抗镍基因相结合的新型分析方法，用于鉴定天蓝遏蓝菜超富集植物中的镍物种。通过尺寸排阻高效液相色谱-毛细管电泳结合电感耦合等离子体质谱检测，证明了一种未知强镍络合物的存在。含镍峰通过电喷雾质谱（质荷比360）进行表征，并通过碰撞诱导解离质谱显示为与三羧酸氨基酸配体形成的螯合物。为了鉴定该物种并证明其功能特性，从天蓝遏蓝菜构建了一个cDNA文库，在酵母中表达，并在有毒的Ni2+培养基上进行筛选。存活转化体培养物的提取物给出了相同的高效液相色谱-电感耦合等离子体质谱、毛细管区带电泳-电感耦合等离子体质谱和电喷雾串联质谱数据，并且含有与烟酰胺合酶基因同源的cDNA插入片段。这一观察结果使得能够鉴定烟酰胺为镍结合配体。通过比较植物和酵母提取物与合成标准品的串联质谱，最终证明了镍-烟酰胺络合物的存在。
