Separation and Identification of Metallothionein Isoforms and Sub-isoforms by Liquid Chromatography
- 化学化工－已发表论文 
建立了金属硫蛋白(MT)异构体及亚型异构体的色谱分离与质谱鉴别方法。将金属硫蛋白混合物通过弱阴离子DEAESephadexA 25离子交换柱,结合离线电感耦合等离子体质谱(ICP MS)对锌诱导金属硫蛋白的两个异构体MT 1和MT 2进行分离和检测;利用SephadexG 25凝胶排阻色谱柱对得到的两个金属硫蛋白异构体进行脱盐;探索脱盐后的金属硫蛋白异构体在不同色谱条件下的C18反相色谱柱上的保留行为,进而实现各个亚型异构体的分离;通过在线电喷雾质谱检测实现了对金属硫蛋白各个亚型异构体的鉴别。结果表明,通过优化色谱条件,由离子交换色谱及凝胶排阻色谱得到的金属硫蛋白各亚型异构体在酸性条件下均得到了良好的分离,质谱检测结果与前人的文献报道结果一致。该方法可使金属硫蛋白各异构体均达到最佳的分离效果。A method has been established for the separation and identification of metallothionein (MT) isoforms and sub-isoforms by liquid chromatography. A mixture of rabbit liver MT was separated on a DEAE A-25 weak anion-exchange chromatographic (AEC) column. Inductively coupled plasma-mass spectrometer (ICP-MS) was used for off-line detection of Zn in each fraction. The two main MT isoforms, MT-1 and MT-2, were isolated. The collected MT isoform fractions were then desalted by a homemade Sephadex G-25 size exclusion chromatographic (SEC) column. Afterwards, the sample was separated on a C_(18) reversed-phase column with UV detection at 210 nm. Different separation conditions were discussed and several sub-isoforms of MT were well separated at pH 2.0. The sub-isoforms were finally characterized by on-line HPLC-ESI-MS. The isoforms and sub-isoforms were well separated on a reversed-phase column under optimized chromatographic conditions and the detection results of ESI-MS were in agreement with the data found in literature. The method developed can be well used for the separation of metallothionein isoforms and sub-isoforms.