Department of Physical and Analytical Chemistry, University of Oviedo, C/Julian Claveria 8, 33006 Oviedo, Spain.
Anal Chem. 2010 Mar 15;82(6):2387-94. doi: 10.1021/ac902624b.
The interest on accurate and precise determination of metalloproteins such as Cu, Zn-superoxide dismutase (Cu, Zn-SOD) involved in the redox balance of living cells is increasing. For this purpose, analytical strategies that provide absolute protein concentration measurements have to be developed. The determination of Cu, Zn-SOD through the measurement of the Cu associated to the protein, which provides its enzymatic activity, by liquid chromatography with online inductively coupled plasma mass spectrometric (ICPMS) detection is described here. Postcolumn isotope dilution analysis (IDA) of Cu has been applied for quantification after evaluation of the column recovery for the total Cu and also Cu-SOD that turned out to be quantitative. When the concentration results obtained via IDA using high-performance liquid chromatography (HPLC)-ICPMS are plotted versus the activity measurements (using the spectrophotometric pyrogallol autoxidation method) a good correlation curve is obtained. Such results permit us, from ICPMS measurements, to obtain simultaneously the Cu, Zn-SOD absolute concentration as well as its enzymatic activity by interpolation in the previously obtained curve. This possibility was explored in real samples (red blood cells of control individuals and patients with metallic total hip arthroplasty) obtaining a good match between direct enzymatic activity measurements and those obtained by interpolation in the correlation curve. The actual protein identification in the red blood cell extract was conducted by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and two matrixes were compared in order to preserve as much as possible the protein-metal interactions during the MALDI process. Interestingly, using a solution containing trihydroxyacetophenone in citrate buffer permitted us to observe some metal-protein interactions in the MS spectrum of the intact Cu, Zn-SOD from red blood cells.
对参与细胞氧化还原平衡的金属蛋白（如 Cu、Zn-超氧化物歧化酶（Cu、Zn-SOD））进行准确和精确测定的兴趣日益增加。为此，必须开发提供绝对蛋白质浓度测量的分析策略。本文描述了通过测量与蛋白质结合的 Cu 来测定 Cu、Zn-SOD 的方法，该方法提供了其酶活性，通过在线电感耦合等离子体质谱（ICPMS）检测的液相色谱法进行。在线后柱同位素稀释分析（IDA）已应用于 Cu 的定量，同时评估了总 Cu 和 Cu-SOD 的柱回收率，结果表明这是定量的。当通过 IDA 使用高效液相色谱（HPLC）-ICPMS 获得的浓度结果与活性测量（使用分光光度法邻苯三酚自氧化法）进行比较时，会得到一条很好的相关曲线。通过这种结果，我们可以从 ICPMS 测量中通过插值获得先前获得的曲线来同时获得 Cu、Zn-SOD 的绝对浓度及其酶活性。在实际样品（对照个体和患有金属全髋关节置换术的患者的红细胞）中探索了这种可能性，直接酶活性测量与通过相关曲线插值获得的测量值之间非常匹配。通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）对红细胞提取物中的实际蛋白质进行鉴定，并比较了两种基质，以便在 MALDI 过程中尽可能多地保留蛋白质-金属相互作用。有趣的是，使用含有三羟基苯乙酮的柠檬酸缓冲液溶液允许我们在完整的 Cu、Zn-SOD 的 MS 光谱中观察到红细胞中的一些金属-蛋白质相互作用。
