Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide.
Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide.
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DOI：
10.3390/molecules26082206
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发表时间：
2021-04-12
期刊：
影响因子：
--
通讯作者：
Guo J
中科院分区：
文献类型：
--
作者：
Pham T;Liao R;Labaer J;Guo J
了解复杂细胞系统的组成、功能和调控需要量化多种蛋白质在其天然细胞环境中表达的工具。在这里，我们报告了一个高度敏感和准确的蛋白质原位分析方法，使用现成的抗体和可切割的荧光酪胺（CFT）。在该方法的每个循环中，用辣根过氧化物酶（HRP）缀合的抗体和CFT对蛋白质靶标进行染色。随后，荧光团被温和的化学试剂有效地切割，同时使HRP失活。通过蛋白质染色、荧光成像、荧光团切割和HRP失活的重复循环，可以实现单细胞原位的多重蛋白质定量。我们设计并合成了高性能的CFT，并证明了超过95%的染色信号可以被温和的化学试剂擦除，同时保留蛋白靶点上表位的完整性。应用这种方法，我们探讨了蛋白质表达的异质性和相关性在一组遗传相同的细胞。由于高信号去除效率，这种方法还使我们能够准确地分析福尔马林固定石蜡包埋（FFPE）组织中的蛋白质，表达水平从低到高，从高到低。
Understanding the composition, function and regulation of complex cellular systems requires tools that quantify the expression of multiple proteins at their native cellular context. Here, we report a highly sensitive and accurate protein in situ profiling approach using off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this method, protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and CFT. Subsequently, the fluorophores are efficiently cleaved by mild chemical reagents, which simultaneously deactivate HRP. Through reiterative cycles of protein staining, fluorescence imaging, fluorophore cleavage, and HRP deactivation, multiplexed protein quantification in single cells in situ can be achieved. We designed and synthesized the high-performance CFT, and demonstrated that over 95% of the staining signals can be erased by mild chemical reagents while preserving the integrity of the epitopes on protein targets. Applying this method, we explored the protein expression heterogeneity and correlation in a group of genetically identical cells. With the high signal removal efficiency, this approach also enables us to accurately profile proteins in formalin-fixed paraffin-embedded (FFPE) tissues in the order of low to high and also high to low expression levels.
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影响因子：
4.8
作者：
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通讯作者：
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30.8
作者：
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通讯作者：
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