Although purification of biotinylated molecules is highly efficient, identifying specific sites of biotinylation remains challenging. We show that anti-biotin antibodies enable unprecedented enrichment of biotinylated peptides from complex peptide mixtures. Live-cell proximity labeling using APEX peroxidase followed by anti-biotin enrichment and mass spectrometry yielded over 1,600 biotinylation sites on hundreds of proteins, an increase of more than 30-fold in the number of biotinylation sites identified compared to streptavidin-based enrichment of proteins.
Protein Data Bank
Portions of this work were supported by HHMI Collaborative Innovation Awards to S.A.C. and A.Y.T. (Norbert Perrimon PI). K.P. was supported by a National Science Foundation Graduate Research Fellowship and a Stanford Graduate Fellowship. T.A. is supported by the Helen Hay Whitney Foundation fellowship. V.K.M. is an Investigator of the Howard Hughes Medical Institute.
Integrated supplementary information
NHS-Biotin Peptide Spike-ins
Comparison of anti-biotin antibody and NeutrAvidin enrichment
Proteins: APEX labelling Streptavidin Enrichment
Biotinylation Sites: APEX labelling Streptavidin Enrichment
Biotinylation Sites: APEX labelling Anti-Biotin Antibody Enrichment; all
Biotinylation Sites: APEX labelling Anti-Biotin Antibody Enrichment; >= 2 replicates
Coverage of OXPHOS proteins