色谱

色谱

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金属硫蛋白异构体及亚型异构体的液相色谱分离与质谱鉴别

卢素格1,沈金灿1,庄峙厦1,2,王小如1,2   

  1. 1. Department of Chemistry and the Key Laboratory of Analytical Science of the Ministry of Education, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China; 2. Qingdao Key Laboratory of Analytical Technology Development and Standardization of Chinese Medicine, the First Institute of Oceanography, State Oceanic Administration, Qingdao 266061, China
  • 收稿日期:2004-03-30 修回日期:1900-01-01 出版日期:2005-03-30 发布日期:1988-12-25
  • 通讯作者: 庄峙厦
  • 基金资助:

Separation and Identification of Metallothionein Isoforms and Sub-isoforms by Liquid Chromatography

LU Suge1,SHEN Jincan1,ZHUANG Zhixia1,2,WANG Xiaoru1,2   

  1. 1. Department of Chemistry and the Key Laboratory of Analytical Science of the Ministry of Education, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China; 2. Qingdao Key Laboratory of Analytical Technology Development and Standardization of Chinese Medicine, the First Institute of Oceanography, State Oceanic Administration, Qingdao 266061, China
  • Received:2004-03-30 Revised:1900-01-01 Online:2005-03-30 Published:1988-12-25

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摘要： 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DEAE Sephadex A-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Sephadex G-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。

关键词: 反相高效液相色谱, 金属硫蛋白, 离子交换色谱, 凝胶排阻色谱, 异构体

Abstract: 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 C18 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.

Key words: gel exclusion chromatography, isoforms , metallothioneins, reversed-phase high performance liquid chromatography, ion-exchange chromatography