通过腺相关病毒介导的近距离标记和质谱法分析小鼠大脑单细胞型蛋白质组

Q4 Biochemistry, Genetics and Molecular Biology Methods in molecular biology Pub Date : 2024-01-01 DOI:10.1007/978-1-0716-3934-4_10
Him K Shrestha, Huan Sun, Ju Wang, Junmin Peng
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引用次数: 0

摘要

单细胞型蛋白质组学是一个新兴的研究领域,它将细胞型特异性与大体蛋白质组学提供的全面蛋白质组覆盖范围相结合。然而,单细胞型蛋白质组的提取仍然是一项挑战,尤其是对于神经元等难以分离的细胞。在本章中,我们介绍了一种利用腺相关病毒(AAV)介导的接近标记(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。这项技术无需进行细胞分离,工作流程简化,包括利用 AAV 表达由细胞特异性启动子控制的 TurboID(一种工程化生物素连接酶)、生物素化蛋白质纯化、珠上消化、TMT 标记和液相色谱-质谱联用(LC-MS)。我们通过分析小鼠不同的脑细胞类型来检验这种方法。首先,使用重组 AAV 在神经元或星形胶质细胞特异性启动子的驱动下同时表达 TurboID 和 mCherry 蛋白,并通过与细胞标记物的联合免疫染色进行验证。通过生物素纯化和TMT分析,我们成功地从几微克的蛋白质样本中鉴定出了约10,000种独特的蛋白质,而且重现性很高。我们的统计分析显示,这些蛋白质组包含细胞类型特异的细胞通路。利用这项技术,研究人员可以探索特定细胞类型的蛋白质组图谱,为深入了解细胞过程、破译疾病机理以及确定神经科学及其他领域的治疗靶点铺平道路。
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Profiling Mouse Brain Single-Cell-Type Proteomes Via Adeno-Associated Virus-Mediated Proximity Labeling and Mass Spectrometry.

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.

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来源期刊
Methods in molecular biology
Methods in molecular biology Biochemistry, Genetics and Molecular Biology-Genetics
CiteScore
2.00
自引率
0.00%
发文量
3536
期刊介绍: For over 20 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice.
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