Separation of metallothionein isoforms extracted from isoform-specific knockdown cells on two-dimensional micro high-performance liquid chromatography hyphenated with inductively coupled plasma-mass spectrometry
Separation of metallothionein isoforms extracted from isoform-specific knockdown cells on two-dimensional micro high-performance liquid chromatography hyphenated with inductively coupled plasma-mass spectrometry
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DOI：
10.1039/b613662c
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发表时间：
2007-02
影响因子：
3.4
通讯作者：
Takamitsu Miyayama;Y. Ogra;Kazuo T. Suzuki
中科院分区：
文献类型：
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作者：
Takamitsu Miyayama;Y. Ogra;Kazuo T. Suzuki
建立了由凝胶过滤柱和阴离子交换柱组成的二维（2D）微型高效液相色谱-电感耦合等离子体质谱（ICP-MS）分离体系，以分离金属硫蛋白（MT）的两种异构体。采用RNA干扰（RNAi）技术特异性敲除MT-I和MT-II基因，并通过逆转录聚合酶链反应（RT-PCR）和2D微高效液相色谱- icp - ms系统评估敲除效果。我们的二维微量HPLC-ICP-MS系统所需的最小细胞数为2.0 × 103， MT蛋白的检出限为22.2 μg L−1。
A two-dimensional (2D) micro HPLC-inductively coupled plasma mass spectrometry (ICP-MS) system consisting of a gel filtration column and an anion-exchange column was constructed to separate two metallothionein (MT) isoforms. MT-I and MT-II genes were specifically knocked down with the RNA interference (RNAi) technique, and the effects of the knockdown were evaluated by the reverse transcription-polymerase chain reaction (RT-PCR) and the 2D micro HPLC-ICP-MS system. The minimum cell number required by our 2D micro HPLC-ICP-MS system was calculated to be 2.0 × 103, and the detection limit of MT protein was estimated to be 22.2 μg L−1.