Characterization of Protein Biotinylation Sites by Peptide-Based Immunoaffinity Enrichment.

Biotin labeling in combination with LC-MS/MS has been widely applied in large-scale analysis of protein post-translational modifications, cell surface proteins, protein-protein interactions, and protein subcellular localization. Direct identification of protein biotinylation sites are still challenging due to the low recovery of biotinylated peptides using conventional streptavidin/avidin-based purification methods. It has been found that anti-biotin antibody is a better capture reagent for biotinylated peptides compared to streptavidin/avidin. In this study, we established an immunoaffinity enrichment method using a monoclonal anti-biotin antibody and compared it to the published approaches from two papers using polyclonal antibodies from various vendors. We then demonstrated our enrichment method by applying it to characterization of protein biotinylation sites from proximity labeling studies in living cells.