BioSITe: A Method for Direct Detection and Quantitation of Site-Specific Biotinylation

Biotin-based labeling strategies
are widely employed to study protein-protein
interactions, subcellular proteomes and post-translational modifications,
as well as, used in drug discovery. While the high affinity of streptavidin
for biotin greatly facilitates the capture of biotinylated proteins,
it still presents a challenge, as currently employed, for the recovery
of biotinylated peptides. Here we describe a strategy designated Biotinylation
Site Identification Technology (BioSITe) for the capture of biotinylated
peptides for LC–MS/MS analyses. We demonstrate the utility
of BioSITe when applied to proximity-dependent labeling methods, APEX
and BioID, as well as biotin-based click chemistry strategies for
identifying O-GlcNAc-modified sites. We demonstrate the use of isotopically
labeled biotin for quantitative BioSITe experiments that simplify
differential interactome analysis and obviate the need for metabolic
labeling strategies such as SILAC. Our data also highlight the potential
value of site-specific biotinylation in providing spatial and topological
information about proteins and protein complexes. Overall, we anticipate
that BioSITe will replace the conventional methods in studies where
detection of biotinylation sites is important.