凝胶限域滚环扩增实现灵敏的单细胞蛋白形式分析

中文摘要 AI 辅助

蛋白质丰度本身并不能反映由蛋白质加工和翻译后修饰所产生的分子多样性，然而大多数单细胞蛋白质检测方法无法解析这些蛋白形式状态。在此，我们开发了RCAmp-scWB，一种分离后扩增策略，在单细胞蛋白质电泳后直接在聚丙烯酰胺凝胶内进行滚环扩增。反应-传输模型确定了试剂可及性与模板关联扩增产物限域之间的平衡，该平衡支持信号放大，同时保留电泳编码的空间读数。使用纯化的蛋白质标准品，RCAmp-scWB产生的浓度-响应斜率比传统单细胞蛋白质印迹陡4.0-7.8倍。在六种人类癌细胞系中，RCAmp-scWB定量了蛋白质丰度，并解析了不同的Vimentin和PD-L1蛋白形式状态，其相对丰度和单细胞分布无法仅通过总蛋白质测量获得。进一步扩展到单个小细胞外囊泡，揭示了Vimentin蛋白形式组成中来源依赖性的变化，尽管一种主要Vimentin物种的丰度相对相似。通过将电泳分子鉴别与信号放大分离，RCAmp-scWB能够对异质细胞和小细胞外囊泡进行蛋白形式分辨的分析。

英文摘要

Protein abundance alone does not capture the molecular diversity generated by protein processing and post-translational modification, yet most single-cell protein assays do not resolve these proteoform states. Here, we develop RCAmp-scWB, a post-separation amplification strategy that performs rolling-circle amplification directly within the polyacrylamide gel after single-cell protein electrophoresis. Reaction-transport modeling identifies a balance between reagent access and confinement of the template-associated amplification product that supports signal amplification while retaining the electrophoretically encoded spatial readout. Using purified protein standards, RCAmp-scWB produced a 4.0-7.8-fold steeper concentration-response slope than conventional single-cell western blotting. Across six human cancer cell lines, RCAmp-scWB quantified protein abundance and resolved distinct Vimentin and PD-L1 proteoform states whose relative abundance and single-cell distributions were not captured by total protein measurements alone. Extension to individual small extracellular vesicles further revealed source-dependent shifts in Vimentin proteoform composition despite comparatively similar abundance of one major Vimentin species. By separating electrophoretic molecular discrimination from signal amplification, RCAmp-scWB enables proteoform-resolved analysis of heterogeneous cells and small extracellular vesicles.

发表机构

• The University of Georgia（佐治亚大学）

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