Summary: The ascorbate peroxidase APEX2 is commonly used to study the neighborhood of a protein of Miniatures Kit interest by proximity-dependent biotinylation.Here, we describe a protocol for sample processing compatible with immunoblotting and mass spectrometry, 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, Nails 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).