Department of Chemistry, University of Kansas, Lawrence, Kansas, USA.
J Sep Sci. 2021 Feb;44(4):895-902. doi: 10.1002/jssc.202000884. Epub 2020 Dec 7.
The calcium signaling protein calmodulin regulates numerous intracellular processes. We introduce a sensitive microchip assay to separate and detect calmodulin binding proteins. The assay utilizes an optimized microchip electrophoresis protein separation platform with laser-induced fluorescence detection. Fluorescence-labeled calmodulin modified with a photoreactive diazirine crosslinker allowed selective detection of calmodulin binding proteins. We demonstrate successful in crosslinking of calmodulin with two calmodulin binding proteins, calcineurin and nitric oxide synthase. We compare the efficacy of commonly applied electrophoretic separation modes: microchip capillary zone electrophoresis, microchip micellar electrokinetic chromatography/gel electrophoresis, and nanoparticle colloidal arrays. Out of the methods tested, polydymethylsiloxane/glass chips with microchip zone electrophoresis gave the poorest separation, whereas sieving methods in which electro-osmotic flow was suppressed gave the best separation of photoproducts of calmodulin conjugated with calmodulin binding proteins.
钙信号蛋白钙调蛋白调节许多细胞内过程。我们引入了一种灵敏的微芯片分析方法来分离和检测钙调蛋白结合蛋白。该分析方法利用了优化的微芯片电泳蛋白质分离平台和激光诱导荧光检测。用光反应性重氮化合物交联剂修饰的荧光标记钙调蛋白允许选择性地检测钙调蛋白结合蛋白。我们成功地将钙调蛋白与两种钙调蛋白结合蛋白（钙调磷酸酶和一氧化氮合酶）交联。我们比较了常用电泳分离模式的效果：微芯片毛细管区带电泳、微芯片胶束电动色谱/凝胶电泳和纳米颗粒胶体阵列。在测试的方法中，聚二甲基硅氧烷/玻璃芯片的微芯片区带电泳分离效果最差，而抑制电渗流的筛分方法则能最好地分离与钙调蛋白结合蛋白偶联的钙调蛋白的光产物。
