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

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 level of biotinylated proteins in cells is low; therefore, large
amounts of cellular proteins were required to detect biotinylated
peptides. In addition, the enriched biotinylated peptide solution
contained many contaminant ions. Here, we optimized the workflow for
efficient enrichment of biotinylated peptides and removal of contaminant
ions. The efficient recovery of biotinylated peptides with fewer contaminant
ions was achieved by heat inactivation of trypsin, prewashing Tamavidin
2-REV beads, clean-up of biotin solution, mock elution, and using
optimal temperature and salt concentration for elution. The optimized
workflow enabled identification of nearly 4-fold more biotinylated
peptides with higher purity from RAW264.7 macrophages expressing TurboID-fused
STING (stimulator of interferon genes). In addition, sequential digestion
with Glu-C and trypsin revealed 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 epidermal growth factor (EGF)
stimulation. This workflow will be useful for BioID and cell surface
proteomics and for various other applications based on protein biotinylation.