Analytical Spectroscopy and Sensors Group, Institute of Environmental Sciences, University of Zaragoza, Pedro Cerbuna, 12 Zaragoza-50009, Spain.
Talanta. 2010 Apr 15;81(1-2):241-7. doi: 10.1016/j.talanta.2009.11.064. Epub 2009 Dec 5.
Some experiments to study the influence of electrophoresis conditions and subsequent LA-ICP-MS (laser ablation-inductively coupled plasma mass spectrometry) determination of two metal-binding proteins with different metal-protein affinities (superoxide dismutase, containing Cu and Zn, and alcohol dehydrogenase, containing Zn) are performed. In metal-binding proteins with weak metal-protein affinities, metal losses can happen during electrophoretic separation. It has been demonstrated that the detection of these metals bound to the proteins depends, not only on the nature of the electrophoretic process (naturing or non-denaturing) and post-separation gel treatment, but also on the trailing ion chosen and current applied in the electrophoretic method used. Non-denaturing methods are preferred to denaturing ones in the case of alcohol dehydrogenase being BN-PAGE (Blue Native-Polyacrylamide Gel Electrophoresis) with the use of Tricine as trailing ion the most recommended method. The concentration obtained for Zn in ADH applying BN-PAGE-LA-ICP-MS was 2.6+/-0.30 mg g(-1) very close to the one obtained for ADH solution by ICP-MS (3+/-0.23 mg g(1)). For superoxide dismutase either denaturing or non-denaturing electrophoresis conditions can be used, but a denaturing method based on the use of Tricine as trailing ion is recommended to preserve metals-protein binding when the use of non-denaturing conditions must be avoided. The found concentration for Cu and Zn in SOD after applying SDS-Tris-Tricine-PAGE-LA-ICP-MS was 2.5+/-0.33 and 2.4+/-0.37 mg g(-1) respectively, more or less close (especially for Cu) to the one obtained in SOD solution by ICP-MS (3+/-0.21 and 3.7+/-0.32 mg g(-1) for Cu and Zn). We observe that as higher current is applied the possibility of metal-protein binding losses is higher. In all cases staining of the gel prior to LA-ICP-MS is not recommended.
进行了一些实验，以研究电泳条件的影响，以及随后使用激光烧蚀电感耦合等离子体质谱法（LA-ICP-MS）测定两种金属结合蛋白（含有 Cu 和 Zn 的超氧化物歧化酶和含有 Zn 的醇脱氢酶）的情况。在金属结合蛋白中，金属蛋白亲和力较弱的情况下，在电泳分离过程中可能会发生金属损失。已经证明，这些与蛋白质结合的金属的检测不仅取决于电泳过程的性质（天然或非变性）和凝胶分离后的处理，还取决于所选择的尾随离子和用于电泳方法的电流。在使用 Tricine 作为尾随离子的 BN-PAGE（蓝色天然聚丙烯酰胺凝胶电泳）情况下，非变性方法优先于变性方法，是醇脱氢酶的最佳方法。应用 BN-PAGE-LA-ICP-MS 测定 ADH 中的 Zn 浓度为 2.6+/-0.30 mg g(-1)，非常接近 ICP-MS 测定 ADH 溶液中的 Zn 浓度（3+/-0.23 mg g(1)）。对于超氧化物歧化酶，既可以使用变性电泳条件，也可以使用非变性电泳条件，但如果必须避免使用非变性条件，则建议使用基于 Tricine 作为尾随离子的变性方法，以保持金属-蛋白质结合。应用 SDS-Tris-Tricine-PAGE-LA-ICP-MS 后，在 SOD 中发现 Cu 和 Zn 的浓度分别为 2.5+/-0.33 和 2.4+/-0.37 mg g(-1)，与 ICP-MS 测定 SOD 溶液中的 Cu 和 Zn 浓度（3+/-0.21 和 3.7+/-0.32 mg g(-1)）相差不大（特别是 Cu）。我们观察到，应用更高的电流时，金属-蛋白质结合损失的可能性更高。在所有情况下，LA-ICP-MS 之前不建议对凝胶进行染色。
