单细胞型蛋白质组学是一个新兴的研究领域,它将细胞类型特异性与批量蛋白质组学提供的全面蛋白质组覆盖相结合。然而,单细胞型蛋白质组的提取仍然是一个挑战,特别是对于像神经元这样难以分离的细胞。在这一章中,我们提出了一种使用腺相关病毒(AAV)介导的邻近标记(PL)和串联质量标签(TMT)质谱分析单细胞型蛋白质组的创新技术。这种技术消除了细胞隔离的需要,并提供了一个简化的工作流程,包括AAV递送以表达由细胞类型特异性启动子控制的TurboID(工程化生物素连接酶),生物素化蛋白纯化,珠上消化,TMT标签,和液相色谱-质谱(LC-MS)。我们通过分析小鼠中不同的脑细胞类型来检查这种方法。最初,重组AAV用于同时表达由神经元或星形胶质细胞特异性启动子驱动的TurboID和mCherry蛋白,通过与细胞标志物的共免疫染色进行验证。用生物素纯化和TMT分析,我们成功地从几微克的蛋白质样品中鉴定出了约10,000种独特的蛋白质,具有很高的可重复性.我们的统计分析显示,这些蛋白质组包含细胞类型特异性细胞通路。通过利用这种技术,研究人员可以探索特定细胞类型的蛋白质组景观,为细胞过程的新见解铺平道路,破译疾病机制,并确定神经科学及其他领域的治疗目标。
Single-cell-type proteomics is an emerging field of research that combines cell-type specificity with the comprehensive proteome coverage offered by bulk proteomics. However, the extraction of single-cell-type proteomes remains a challenge, particularly for hard-to-isolate cells like neurons. In this chapter, we present an innovative technique for profiling single-cell-type proteomes using adeno-associated virus (AAV)-mediated proximity labeling (PL) and tandem-mass-tag (TMT) mass spectrometry. This technique eliminates the need for cell isolation and offers a streamlined workflow, including AAV delivery to express
TurboID (an engineered biotin ligase) controlled by cell-type-specific promoters, biotinylated protein purification, on-bead digestion, TMT labeling, and liquid chromatography-mass spectrometry (LC-MS). We examined this method by analyzing distinct brain cell types in mice. Initially, recombinant AAVs were used to concurrently express
TurboID and mCherry proteins driven by neuron- or astrocyte-specific promoters, which was validated through co-immunostaining with cellular markers. With biotin purification and TMT analysis, we successfully identified around 10,000 unique proteins from a few micrograms of protein samples with high reproducibility. Our statistical analyses revealed that these proteomes encompass cell-type-specific cellular pathways. By utilizing this technique, researchers can explore the proteomic landscape of specific cell types, paving the way for new insights into cellular processes, deciphering disease mechanisms, and identifying therapeutic targets in neuroscience and beyond.