Optimized Workflow for Enrichment and Identification of Biotinylated Peptides using Tamavidin 2-REV for BioID and Cell Surface Proteomics

Abstract

Chemical or enzymatic biotinylation of proteins is widely used in various studies, and proximity-dependent biotinylation coupled to mass spectrometry is a powerful approach for analyzing protein–protein interactions in living cells. We recently developed a simple method to enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. However, the low abundance of protein biotinylation in cells required large amounts of cellular proteins to detect enough biotinylated peptides. Here we optimized the workflow for efficient enrichment and identification of biotinylated peptides. The most efficient recovery was achieved by heat inactivation of trypsin, prewashing Tamavidin 2-REV beads, clean-up of biotin solution, mock elution, and the optimal temperature and salt concentration for elution. Using the optimized workflow, over 2-fold more biotinylated peptides were identified with higher purity from RAW264.7 macrophages expressing TurboID-fused STING. In addition, sequential digestion with Glu-C and trypsin led to the identification of biotinylation sites that were not identified by trypsin digestion alone. Furthermore, the combination of this workflow with TMT labeling enabled large-scale quantification of cell surface proteome changes upon EGF stimulation. This workflow would be useful not only for BioID and cell surface proteomics but also for various other applications based on protein biotinylation.