Soto Joselyn S, Khakh Baljit S
Department of Physiology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA.
Department of Biological Science, California State University Fullerton, Fullerton, CA, USA.
Methods Mol Biol. 2025;2953:281-294. doi: 10.1007/978-1-0716-4694-6_18.
Neurons and astrocytes interact in the brain to form a complex network with physical, chemical, and electrical connectivity. Traditional methods for investigating proteomes have primarily employed either primary cell cultures or cells acutely separated from tissue and purified based on cell surface markers. However, these methods often compromise the cellular physiological integrity, leading to loss of associated proteins and native connectivity. Therefore, to understand the role of astrocytes and their interactions with neurons at the protein level, we designed a cell-specific method using the biotin ligase BioID2, which enables tagging of proteomes in distinct subcellular compartments of astrocytes and neurons in vivo. Here, we describe step-by-step protocols for the in vivo expression of BioID2, followed by biotinylation and subsequent purification of biotinylated proteins for mass spectrometry analysis using a brain-wide adeno-associated viral (AAV) approach.
神经元和星形胶质细胞在大脑中相互作用，形成一个具有物理、化学和电连接性的复杂网络。传统的蛋白质组研究方法主要采用原代细胞培养或从组织中急性分离并基于细胞表面标志物纯化的细胞。然而，这些方法常常会破坏细胞的生理完整性，导致相关蛋白质的丢失和天然连接性的丧失。因此，为了在蛋白质水平上理解星形胶质细胞的作用及其与神经元的相互作用，我们设计了一种利用生物素连接酶BioID2的细胞特异性方法，该方法能够在体内对星形胶质细胞和神经元不同亚细胞区室中的蛋白质组进行标记。在此，我们描述了BioID2在体内表达、随后进行生物素化以及随后纯化生物素化蛋白质以用于使用全脑腺相关病毒（AAV）方法进行质谱分析的分步方案。
