Detection and Quantification of Biotinylated Sites for TurboID-Based Proximity Labeling Mass Spectrometry in...

Proximity labeling mass spectrometry (PL-MS) is a powerful technique for mapping protein-protein interactions (PPIs), subcellular and cell type-specific proteomes, and protein-RNA and protein-DNA interactions. Direct detection of biotinylated peptides is critical for the accurate characterization of proximity-tagged proteins. A peptide-level enrichment workflow is advantageous as it facilitates the efficient removal of free biotin prior to enrichment, thus allowing for the use of higher concentrations of biotin-an important advantage in certain plant studies. This chapter provides a detailed, step-by-step protocol for sample preparation, including enrichment methods using both anti-biotin antibody-based and streptavidin-based affinity beads. Additionally, it offers an overview of software tools crucial for the reliable identification and quantification of biotinylated peptides.