Zhai Xinke, Cao Yiren, Tian Youli, Chen Ke-Er, Yu Zixian, Zhang Qiang, Cao Chengxi, Xu Xu
School of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, China.
School of Automation and Sensing, Shanghai Jiao Tong University, Shanghai, 200240, China.
Anal Methods. 2025 Jul 10;17(27):5749-5758. doi: 10.1039/d4ay02064b.
Traditional polyacrylamide gel electrophoresis (PAGE) has been widely used for analysis of proteins. However, protein quantification (PQ) based on SDS-PAGE with staining remains inaccurate, time-consuming and complex. Even though PAGE with online intrinsic fluorescence imaging (IFI) has well addressed these issues, it still suffers from the narrow detection window of 8 lanes and poor quantitation owing to low resolution. Herein, we improved the gel electrophoresis tank (GET) for a wider detection window of 10 lanes with uniform UV radiation and developed a Gaussian fitting arithmetic for accurate PQ at low resolutions. To demonstrate the improvement, we used bovine serum albumin (BSA) in whey samples as a model protein and conducted the relevant experiments. As demonstrated by the experiments, the IFI system could be well used for the simultaneous real-time imaging analysis of 10 lanes. In particular, the Gaussian fitting-based analyzer could be used for the computation of peak area with low resolution, greatly improving the function of PAGE-IFI for protein analysis of complex samples. In addition, the developed method exhibited a good limit of detection (LOD) of 14 ng and limit of quantification (LOQ) of 42 ng, a fair dynamic range of 50-8000 ng, recovery of 106.37% for a urine sample, recovery of 94.96% for the whey sample, intra-day RSD value of 9.17%, and inter-day RSD value of 10.06%. All these results indicated the potential of the developed GET, Gaussian fitting-based analyzer and the proposed method for the determination of protein in complex samples.
传统的聚丙烯酰胺凝胶电泳（PAGE）已被广泛用于蛋白质分析。然而，基于SDS-PAGE染色的蛋白质定量（PQ）仍然不准确、耗时且复杂。尽管具有在线固有荧光成像（IFI）的PAGE很好地解决了这些问题，但由于分辨率低，它仍然存在8个泳道的检测窗口狭窄和定量效果差的问题。在此，我们改进了凝胶电泳槽（GET），使其具有10个泳道的更宽检测窗口和均匀的紫外线辐射，并开发了一种高斯拟合算法，用于在低分辨率下进行准确的PQ。为了证明这种改进，我们使用乳清样品中的牛血清白蛋白（BSA）作为模型蛋白并进行了相关实验。实验表明，IFI系统可很好地用于10个泳道的同步实时成像分析。特别是，基于高斯拟合的分析仪可用于低分辨率下峰面积的计算，大大提高了PAGE-IFI对复杂样品蛋白质分析的功能。此外，所开发的方法表现出良好的检测限（LOD）为14 ng，定量限（LOQ）为42 ng，动态范围为50 - 8000 ng，尿液样品回收率为106.37%，乳清样品回收率为94.96%，日内相对标准偏差值为9.17%，日间相对标准偏差值为10.06%。所有这些结果表明所开发的GET、基于高斯拟合的分析仪和所提出的方法在测定复杂样品中蛋白质方面的潜力。
