与微量元素结合的金属蛋白在生物过程中起着至关重要的作用,与外源重金属结合有不利影响。然而,快速的方法,仍然缺乏对这些金属蛋白的高灵敏度和同时分析。在这项研究中,通过将尺寸排阻色谱(SEC)与电感耦合等离子体串联质谱(ICP-MS/MS)相结合,开发了一种同时测定必需和有毒含金属蛋白的快速方法。优化分离和检测条件后,在10分钟内成功分离出7种不同分子量的金属蛋白(从16.0到443.0kDa),含铁(Fe)的蛋白质,铜(Cu),锌(Zn),在ICP-MS/MS中,使用氧气作为碰撞气体可以同时检测碘(I)和铅(Pb)元素。因此,建立对数分子量与保留时间之间的线性关系,以估计未知蛋白质的分子量。因此,可以在单次运行中检测到痕量金属和含有毒金属的蛋白质,并且具有很高的灵敏度(检出限在0.0020-2.5μg/mL范围内)和良好的重复性(相对标准偏差低于4.5%)。该方法随后被成功地用于分析金属(例如,Pb,Zn,铅中毒患者血液中的Cu和Fe)结合蛋白,结果显示锌和铅结合蛋白的含量呈负相关,被鉴定为含有血红蛋白亚基。总之,这项工作为筛选大量生物样品中的含金属蛋白质提供了一种快速灵敏的工具。
Metalloproteins binding with trace elements play a crucial role in biological processes and on the contrary, those binding with exogenous heavy metals have adverse effects. However, the methods for rapid, high sensitivity and simultaneous analysis of these
metalloproteins are still lacking. In this study, a fast method for simultaneously determination of both essential and toxic metal-containing proteins was developed by coupling size exclusion chromatography (SEC) with inductively coupled plasma tandem mass spectrometry (ICP-MS/MS). After optimization of the separation and detection conditions, seven
metalloproteins with different molecular weight (from 16.0 to 443.0 kDa) were successfully separated within 10 min and the proteins containing iron (Fe), copper (Cu), zinc (Zn), iodine (I) and lead (Pb) elements could be simultaneously detected with the use of oxygen as the collision gas in ICP-MS/MS. Accordingly, the linear relationship between log molecular weight and retention time was established to estimate the molecular weight of unknown proteins. Thus, the trace metal and toxic metal containing proteins could be detected in a single run with high sensitivity (detection limits in the range of 0.0020-2.5 μg/mL) and good repeatability (relative standard deviations lower than 4.5 %). This method was then successfully used to analyze metal (e.g., Pb, Zn, Cu and Fe) binding proteins in the blood of Pb-intoxicated patients, and the results showed a negative correlation between the contents of zinc and lead binding proteins, which was identified to contain hemoglobin subunit. In summary, this work provided a rapid and sensitive tool for screening metal containing proteins in large number of biological samples.