Uppsala University, Dept. of Immunology, Genetics and Pathology, Science for Life Laboratory, Biomedical Center, 751 08 Uppsala (JY, AZ, MKM, UL), Rudbeck Laboratory 751 85, Uppsala, (NP, LCW), Sweden.
Department of Biomedical Engineering, Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, 5600, MB, The Netherlands.
Sci Rep. 2017 May 4;7(1):1490. doi: 10.1038/s41598-017-01516-7.
Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms with modest sample input. However, insufficient sensitivity prevents detection of proteins present at low concentrations and antibody cross-reactivity results in unspecific detection that cannot be distinguished from bona fide protein isoforms. By using DNA-conjugated antibodies enhanced signals can be obtained via rolling circle amplification (RCA). Both sensitivity and specificity can be greatly improved in assays dependent on target recognition by pairs of antibodies using in situ proximity ligation assays (PLA). Here we applied these DNA-assisted RCA techniques in capillary isoelectric focusing to resolve endogenous signaling transducers and isoforms along vascular endothelial growth factor (VEGF) signaling pathways at concentrations too low to be detected in standard assays. We also demonstrate background rejection and enhanced specificity when protein detection depended on binding by pairs of antibodies using in situ PLA, compared to assays where each antibody preparation was used on its own.
检测和定量蛋白质及其翻译后修饰对于解析细胞生物学和医学中复杂蛋白质网络的功能至关重要。毛细管等电聚焦与基于抗体的检测相结合，可用于分离和鉴定具有中等样本输入量的蛋白质及其同工型。然而，由于灵敏度不足，该方法无法检测低浓度的蛋白质，并且抗体的交叉反应会导致非特异性检测，无法与真正的蛋白质同工型区分开来。通过使用 DNA 偶联抗体，可通过滚环扩增（RCA）获得增强的信号。利用原位邻近连接分析（PLA）检测依赖于配对抗体的目标识别的分析中，灵敏度和特异性均可得到极大提高。在这里，我们将这些 DNA 辅助的 RCA 技术应用于毛细管等电聚焦中，以解析血管内皮生长因子（VEGF）信号通路中内源性信号转导子和同工型，其浓度低至在标准分析中无法检测到的水平。与每个抗体制剂单独使用的分析相比，当蛋白质检测依赖于原位 PLA 中配对抗体的结合时，我们还证明了背景排斥和增强的特异性。
