Autophagosome content profiling using proximity biotinylation proteomics coupled to protease digestion in mammalian cells

The ascorbate peroxidase APEX2 is commonly used to study the neighborhood of a protein of interest by proximity-dependent biotinylation. Here, we describe a protocol for sample processing compatible with immunoblotting and mass spectrometry that is suitable to specifically map the content of autophagosomes and potentially other short-lived endomembrane transport vesicles without the need of subcellular fractionation. By combining live-cell biotinylation with proteinase K digestion of cell homogenates, proteins enriched in membrane-protected compartments can be readily enriched and identified. For complete details on the use and execution of this protocol, please refer to (Zellner et al., 2021).抗坏血酸过氧化物酶APEX2（ascorbate peroxidase APEX2）常被用于通过邻近依赖生物素标记法（proximity-dependent biotinylation）研究目标蛋白的邻域环境。本文描述了一种兼容免疫印迹法（immunoblotting）与质谱分析法（mass spectrometry）的样本处理流程，该方法无需亚细胞分级分离（subcellular fractionation），即可特异性定位自噬体（autophagosomes）及其他短寿命内膜运输囊泡（endomembrane transport vesicles）的组分。通过将活细胞生物素标记与细胞匀浆（cell homogenates）的蛋白酶K（proteinase K）消化相结合，富集于膜保护区室中的蛋白质可被高效富集并鉴定。如需了解该实验方案的完整使用与操作细节，请参考(Zellner等人，2021)。