We introduce multiplexed single-molecule pull-down and co-immunoprecipitation, named m-SMPC, an analysis tool for profiling multiple protein complexes within a single reaction chamber using single-molecule fluorescence imaging. We employed site-selective conjugation of biotin and fluorescent dye directly onto the monoclonal antibodies, which completed an independent sandwich immunoassay without the issue of host cross-reactivity. We applied this technique to profile endogenous B-cell lymphoma extra-large (BCLxL) complexes in non-small cell lung cancer (NSCLC) cells. Up to three distinct BCLxL complexes were successfully detected simultaneously within a single reaction chamber without fluorescence signal crosstalk. Notably, the NSCLC cell line EBC-1 exhibited high BCLxL-BAX and BCLxL-BAK levels, which closely paralleled a strong response to the BCLxL inhibitor A-1331852. This streamlined method offers the potential for quantitative biomarkers derived from protein complex profiling, paving the way for their application in protein complex-targeted therapies.

中文翻译：

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通过多重单分子 Pull-Down 和免疫共沉淀分析非小细胞肺癌细胞中的 BCLxL 蛋白复合物


我们引入了多重单分子下拉和免疫共沉淀，称为 m-SMPC，这是一种使用单分子荧光成像在单个反应室内分析多个蛋白质复合物的分析工具。我们将生物素和荧光染料直接位点选择性缀合到单克隆抗体上，完成了独立的夹心免疫测定，没有宿主交叉反应的问题。我们应用该技术来分析非小细胞肺癌 (NSCLC) 细胞中的内源性 B 细胞淋巴瘤超大 (BCLxL) 复合物。在单个反应室内可同时成功检测到多达三种不同的 BCLxL 复合物，且没有荧光信号串扰。值得注意的是，NSCLC 细胞系 EBC-1 表现出高 BCLxL-BAX 和 BCLxL-BAK 水平，这与 BCLxL 抑制剂 A-1331852 的强烈反应密切相关。这种简化的方法为从蛋白质复合物分析中衍生出定量生物标志物提供了潜力，为其在蛋白质复合物靶向治疗中的应用铺平了道路。