Characterization Method for Metallothioneins by Reversed- Phase Liquid Chromatography Coupled with Electrospray Ionization Mass Spectrometry
- 化学化工－已发表论文 
通过反相液相色谱(RPLC)与电喷雾电离质谱(ESI MS)的联用技术,对镉诱导金属硫蛋白标准物质MT 1和MT 2的结构进行表征分析。采用VydacC8反相色谱柱(250mm×2 1mmi d ,5μm,30nm),流动相A为pH6 0的5mmol/L乙酸铵水溶液,流动相B为pH6 0的5mmol/L乙酸铵的甲醇 水(体积比为1∶1)溶液,流动相流速为0 20mL/min,在40min内流动相B的体积分数从10%增加到37 5%进行梯度洗脱。分别用紫外(UV)和ESI MS检测,可获得总离子流色谱图及与其色谱峰对应的质谱图。通过对MT 1和MT 2的质谱信号的解析,发现通过该法获得的MT结构形态大多与前人报道的结果吻合。还测定了兔肝MT的多形态结构,利用酸化脱金属硫蛋白(apo MT)使谱图简化,可清晰地获得在兔肝中存在的各种MT亚型和次亚型结构形态。Mammalian metallothioneins (MTs) usually have two major isoforms, MT-1 and MT-2, existing in tissues because of the genetic polymorphism. Metallothionein isoforms and sub-isoforms were characterized with a hyphenated technique of reversed-phase liquid chromatography (RPLC) coupled with electrospray ionization mass spectrometry (ESI-MS). The MT-isoform separation was carried out using a C_8 narrow-bore column Vydac C_8 (250 mm×2.1 mm i.d., 5 μm, 30 nm). Buffer A was 5 (mmol/L) ammonium acetate in water (pH 6.0) and buffer B was 5 (mmol/L) ammonium acetate in methanol-water (1∶1, v/v) (pH 6.0). A linear gradient elution was used by increasing buffer B from 10% to 37.5% within 40 min. The flow rate was set at 0.20 (mL/min). The tentative formulas of MT isoforms and subisoforms were interpreted with the measured masses at the vicinity of apexes in total ion current chromatogram of ESI-MS and the reference data reported previously. The method was validated with the standard reference materials of MT-1 and MT-2. The results indicated that many MT species interpreted were in good agreement with the reference data reported. The MT sample from rabbit liver was measured with the established method. The results, of which the apo-MT was used by (acidification) of the sample for simplifying the mass spectra, showed that there were species of MT isoforms and subisoforms in rabbit livers.