Ryu Taekyung, Kim Seok-Young, Thuraisamy Thujitha, Jang Yura, Na Chan Hyun
Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Neuroregeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
bioRxiv. 2023 Jun 14:2023.06.13.544682. doi: 10.1101/2023.06.13.544682.
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 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.
由于蛋白质是发挥细胞功能的必需分子，解读蛋白质组变化是理解各种疾病正常生理和发病机制的关键。然而，传统蛋白质组学研究通常在组织块上进行，其中多种细胞类型相互缠绕，这给解读不同细胞类型之间的生物学动态带来了挑战。虽然最近出现了一些细胞特异性蛋白质组分析技术，如BONCAT、TurboID和APEX，但它们对基因改造的需求限制了其应用。另一种方法激光捕获显微切割（LCM），虽然它不需要基因改造，但劳动强度大、耗时且需要专业知识，不太适合大规模研究。在本研究中，我们开发了一种使用抗体介导的生物素化（iCAB）进行细胞类型特异性蛋白质组分析的方法，我们将免疫组织化学（IHC）与生物素酪胺信号放大方法相结合。与二抗偶联的多辣根过氧化物酶（HRP）将通过针对目标细胞类型的一抗定位在目标细胞类型上，HRP激活的生物素酪胺将使附近的蛋白质生物素化。因此，iCAB方法可应用于任何可用于IHC的组织。作为概念验证，我们将iCAB应用于小鼠脑组织，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。我们总共从富集和未富集的样本中鉴定出约8400种和约6200种蛋白质。当我们比较不同细胞类型数据时，富集样本中的大多数蛋白质表现出差异表达，而未富集样本中没有差异表达的蛋白质。使用Azimuth对各细胞类型中增加的蛋白质进行细胞类型富集分析表明，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/血管周围巨噬细胞为代表性细胞类型。富集蛋白质的蛋白质组数据显示出与未富集蛋白质相似的亚细胞分布，表明iCAB蛋白质组不偏向任何亚细胞区室。据我们所知，本研究代表了使用抗体介导的生物素化方法进行细胞类型特异性蛋白质组分析方法的首次应用。这一进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，这可以加速我们对生物学和病理现象的理解。
