Lin Zhi, Schaefer Kaitlin, Lui Irene, Yao Zi, Fossati Andrea, Swaney Danielle L, Palar Ajikarunia, Sali Andrej, Wells James A
bioRxiv. 2023 Oct 29:2023.10.28.564055. doi: 10.1101/2023.10.28.564055.
The cell membrane proteome is the primary biohub for cell communication, yet we are only beginning to understand the dynamic protein neighborhoods that form on the cell surface and between cells. Proximity labeling proteomics (PLP) strategies using chemically reactive probes are powerful approaches to yield snapshots of protein neighborhoods but are currently limited to one single resolution based on the probe labeling radius. Here, we describe a multi-scale PLP method with tunable resolution using a commercially available histological dye, Eosin Y, which upon visible light illumination, activates three different photo-probes with labeling radii ranging from ∼100 to 3000 Å. We applied this platform to profile neighborhoods of the oncogenic epidermal growth factor receptor (EGFR) and orthogonally validated >20 neighbors using immuno-assays and AlphaFold-Multimer prediction that generated plausible binary interaction models. We further profiled the protein neighborhoods of cell-cell synapses induced by bi-specific T-cell engagers (BiTEs) and chimeric antigen receptor (CAR)T cells at longer length scales. This integrated multi-scale PLP platform maps local and distal protein networks on cell surfaces and between cells. We believe this information will aid in the systematic construction of the cell surface interactome and reveal new opportunities for immunotherapeutics.
细胞膜蛋白质组是细胞通讯的主要生物枢纽，但我们才刚刚开始了解在细胞表面以及细胞之间形成的动态蛋白质邻域。使用化学反应性探针的邻近标记蛋白质组学（PLP）策略是获取蛋白质邻域快照的有力方法，但目前基于探针标记半径仅限于单一分辨率。在此，我们描述了一种使用市售组织学染料伊红Y的具有可调分辨率的多尺度PLP方法，该染料在可见光照射下会激活三种不同的光探针，其标记半径范围从约100到3000 Å。我们应用该平台对致癌表皮生长因子受体（EGFR）的邻域进行分析，并使用免疫测定和AlphaFold-Multimer预测对20多个邻域进行了正交验证，从而生成了合理的二元相互作用模型。我们进一步在更长的长度尺度上分析了双特异性T细胞衔接器（BiTEs）和嵌合抗原受体（CAR）T细胞诱导的细胞间突触的蛋白质邻域。这个集成的多尺度PLP平台绘制了细胞表面以及细胞之间的局部和远端蛋白质网络。我们相信这些信息将有助于系统构建细胞表面相互作用组，并揭示免疫治疗的新机会。
