The UC Berkeley-UCSF Graduate Program in Bioengineering, University of California, Berkeley, California 94720, United States.
Anal Chem. 2013 Mar 5;85(5):2882-90. doi: 10.1021/ac3035053. Epub 2013 Feb 15.
Post-translational modifications play a critical role in regulating protein function. Increasingly, determination of protein identity, estimation of abundance, and characterization of post-translational modifications are required for analysis of protein-mediated cell signaling networks. As such, we report an integrated and rapid multispectral immunoprobed isoelectric focusing technique for identifying specific proteins bearing post-translational modifications. Immunoprobed isoelectric focusing is composed of isoelectric focusing in a large pore-size polyacrylamide gel to determine protein pI followed by immobilization of pI-resolved proteins. Proteins are immobilized via covalent attachment to a channel-filling benzophenone-functionalized polyacrylamide gel via brief UV exposure (photoblot), followed by multispectral antibody-based detection. The assay correlates observed post-translational modifications to pI shifts relative to the unmodified protein of interest. During the electrokinetically driven antibody probing stage, we observed nonuniform electrophoretic probe mobility along the channel axis. The spatially varying mobility is attributed to nonuniform charge arising from covalent attachment of ampholytes to the benzophenone-functionalized gel matrix during the photoblotting step. Using the multistep microfluidic assay, phosphorylated and acetylated forms of heat shock protein 27 and superoxide dismutase 2 were detected, respectively. The assay reported protein isoforms in immune-purified sample and raw cell lysate in 2 hours with sample volume requirements of 2 μL. This new assay is well-matched to systems biology frameworks for study of protein post-translational modifications.
翻译后修饰在调节蛋白质功能方面起着关键作用。越来越多的情况下，需要确定蛋白质的身份、估计丰度，并对翻译后修饰进行特征描述，以分析蛋白质介导的细胞信号转导网络。因此，我们报告了一种集成的、快速的多光谱免疫探测等电聚焦技术，用于鉴定具有翻译后修饰的特定蛋白质。免疫探测等电聚焦由大孔径聚丙烯酰胺凝胶中的等电聚焦组成，用于确定蛋白质的等电点，然后固定等电聚焦分离的蛋白质。蛋白质通过短时间的紫外光暴露（光印迹）共价附着到填充通道的苯并二酮功能化聚丙烯酰胺凝胶上进行固定，然后通过多光谱抗体检测。该测定法将观察到的翻译后修饰与感兴趣的未修饰蛋白质的等电点变化相关联。在电动力学驱动的抗体探测阶段，我们观察到通道轴上的电泳探针迁移率不均匀。这种空间变化的迁移率归因于在光印迹步骤中，两性电解质与苯并二酮功能化凝胶基质共价结合产生的非均匀电荷。使用多步微流控测定法，分别检测到磷酸化和乙酰化形式的热休克蛋白 27 和超氧化物歧化酶 2。该测定法在 2 小时内，使用 2 μL 的样品体积，可在免疫纯化样品和原始细胞裂解物中检测到蛋白质同工型。这种新测定法非常适合用于研究蛋白质翻译后修饰的系统生物学框架。
