Recently, proximity labeling has been developed to map spatially localized proteomes in live cells. Usually, these methods employ enzymatic biotinylation of the proximal proteins with reactive biotin species. The labeled proteins may contain biotinylated modifications, which can be enriched by streptavidin beads through affinity purification. However, during the bead enrichment process, unlabeled proteins can be enriched to have specific binding affinity toward the biotinylated proteins or high binding affinity to the bead surface. If the unlabeled proteins remain attached to the beads after washing and are analyzed by mass spectrometry (MS) using the conventional workflow for the unlabeled peptidome, they would appear as proximal proteins in the targeted space. However, the unlabeled proteins, including the specific interaction partners of the biotinylated proteins, are false positives for proximity labeling. Including the unlabeled proteome in the identification list for proximity labeling does not provide a clear picture of the local proteome in the targeted space. This chapter is a detailed protocol of the first direct identification method (Spot-BioID) for identifying biotin-labeled proteomes of promiscuous biotin ligase (pBirA) labeling.
Keywords: Biotin-labeled peptide; Engineered ascorbate peroxidase (APEX); Mass spectrometry (MS); Promiscuous biotin ligase (pBirA); Proximity labeling; Spot-ID.