Development of an in situ cell-type specific proteome analysis method using antibody-mediated biotinylation.
Development of an in situ cell-type specific proteome analysis method using antibody-mediated biotinylation.
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使用抗体介导的生物素化开发原位细胞类型特异性蛋白质组分析方法。
DOI：
10.1101/2023.06.13.544682
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Na,ChanHyun
中科院分区：
文献类型：
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作者：
Ryu,Taekyung;Kim,Seok-Young;Thuraisamy,Thujitha;Jang,Yura;Na,ChanHyun
由于蛋白质是发挥细胞功能的重要分子，解码蛋白质组变化是理解各种疾病的正常生理和发病机制的关键。然而，传统的蛋白质组学研究通常是在组织肿块上进行的，其中多种细胞类型纠缠在一起，这对解释不同细胞类型之间的生物学动力学提出了挑战。虽然最近的细胞特异性蛋白质组分析技术，如BONCAT，TurboID和APEX，已经出现，但它们对遗传修饰的必要性限制了它们的使用。另一种方法是激光捕获显微切割（LCM），尽管它不需要遗传改变，但劳动密集型，耗时，需要专业知识，因此不太适合大规模研究。在这项研究中，我们开发了原位细胞类型特异性蛋白质组分析的方法，使用抗体介导的生物素化（iCAB），其中我们结合免疫组织化学（IHC）与生物素-酪胺信号放大方法。与二抗偶联的聚辣根过氧化物酶（HRP）将通过对靶细胞类型具有特异性的一抗定位于靶细胞类型，并且由HRP激活的生物素-酪胺将使附近的蛋白质生物素化。因此，iCAB方法可应用于可用于IHC的任何组织。作为概念验证，我们采用iCAB用于小鼠脑组织，富集神经元细胞体、星形胶质细胞和小胶质细胞的蛋白质，然后使用基于16重TMT的蛋白质组学鉴定富集的蛋白质。我们总共从富集和非富集样品中鉴定了约8，400和约6，200种蛋白质。当我们比较不同细胞类型数据时，富集样品中的大多数蛋白质显示差异表达，而非富集样品中没有差异表达的蛋白质。使用Azimuth进行的各细胞类型中蛋白质增加的细胞类型富集分析显示，神经元细胞体、星形胶质细胞和小胶质细胞数据分别显示谷氨酸能神经元、星形胶质细胞和小胶质细胞/血管周巨噬细胞为代表性细胞类型。富集蛋白的蛋白质组数据显示与非富集蛋白相似的亚细胞分布，表明iCAB-蛋白质组不偏向于任何亚细胞区室。据我们所知，这项研究代表了第一次实施的细胞类型特异性蛋白质组分析方法，使用抗体介导的生物素化方法。这一发展为细胞类型特异性蛋白质组分析的常规和广泛应用铺平了道路。最终，这可能会加速我们对生物和病理现象的理解。
Since proteins are essential molecules exerting cellular functions, decoding proteome changes is the key to understanding the normal physiology and pathogenesis mechanism of various diseases. However, conventional proteomic studies are often conducted on tissue lumps, in which multiple cell types are entangled, presenting challenges in interpreting the biological dynamics among diverse cell types. While recent cell-specific proteome analysis techniques, like BONCAT, TurboID, and APEX, have emerged, their necessity for genetic modifications limits their usage. The alternative, laser capture microdissection (LCM), although it does not require genetic alterations, is labor-intensive, time-consuming, and requires specialized expertise, making it less suitable for large-scale studies. In this study, we develop the method for in situ cell-type specific proteome analysis using antibody-mediated biotinylation (iCAB), in which we combined immunohistochemistry (IHC) with the biotin-tyramide signal amplification approach. Poly-horseradish peroxidase (HRP) conjugated to the secondary antibody will be localized at a target cell type via a primary antibody specific to the target cell type and biotin-tyramide activated by HRP will biotinylate the nearby proteins. Therefore, the iCAB method can be applied to any tissues that can be used for IHC. As a proof-of-concept, we employed iCAB for mouse brain tissue enriching proteins for neuronal cell bodies, astrocytes, and microglia, followed by identifying the enriched proteins using 16-plex TMT-based proteomics. In total, we identified ~8,400 and ~6,200 proteins from enriched and non-enriched samples. Most proteins from the enriched samples showed differential expressions when we compared different cell type data, while there were no differentially expressed proteins from non-enriched samples. The cell type enrichment analysis with the increased proteins in respective cell types using Azimuth showed that neuronal cell bodies, astrocytes, and microglia data exhibited Glutamatergic Neuron, Astrocyte and Microglia/Perivascular Macrophage as the representative cell types, respectively. The proteome data of the enriched proteins showed similar subcellular distribution as non-enriched proteins, indicating that the iCAB-proteome is not biased toward any subcellular compartment. To our best knowledge, this study represents the first implementation of a cell-type-specific proteome analysis method using an antibody-mediated biotinylation approach. This development paves the way for the routine and widespread use of cell-type-specific proteome analysis. Ultimately, this could accelerate our understanding of biological and pathological phenomena.