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.
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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.
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Integrated supplementary information
Supplementary Figure 1 Enrichment with anti-biotin antibody significantly increases detection of biotinylated peptides in complex samples.
(A) Structure of NHS-biotin (B) Results of spike-in studies. Bar plot shows the number of unique biotinylated peptides identified at each biotinylated:non-biotinylated peptide spike-in level. Error bars indicate standard deviation across three replicates of enrichment (C) Results of antibody titration experiments. Plot shows the number of distinct biotinylated peptides (red) and the number of distinct non-biotinylated peptides (black) from enrichment of 1 mg of peptides from 1:1000 spike-in samples. Error bars indicate range across two replicates of enrichment. (D) Results of testing different vendor antibodies.
(A) Anti V5- (top) and and anti-streptavidin (bottom) western blots of SILAC HEK 293T cells labeled with mitochondrial matrix-APEX and biotin phenol. A merged image is also shown. (B) Confocal fluorescence imaging of mitochondrial matrix-APEX2 labeling. Human embryonic kidney (HEK) 293T cells stably expressing matrix-APEX2 were incubated with biotin-phenol (BP) for 30 min and then treated live for 1 min with 1 mM H2O2 to initiate biotinylation (top row). A sample in which both BP and H2O2 were omitted was prepared in parallel as a negative control (bottom row). Cells were fixed and then stained with a NeutrAvidin-Alexa Fluor 647 (AF647) conjugate to visualize biotinylated proteins and an anti-V5 antibody to visualize matrix-APEX2 localization. DIC, differential interference contrast image. White horizontal scale bars = 25 μm.
Supplementary Figure 3 Analysis of SILAC data from the mitochondrial matrix streptavidin enriched proteomic experiments.
(A) Correlation between SILAC log2 H/L ratios for replicates 1, 2 and 3. A high correlation is seen, as expected. R^2 values are shown for all detected proteins. (B) Cutoff determination for the mitochondrial matrix streptavidin enriched proteomic experiment. Histograms (left panel) plot true positives (TP) in green, and false positives (FP) in red. Receiver-operating characteristic analysis (ROC, right panel) was performed for each replicate, setting the SILAC ratio cutoff value such that true positives were maximized and false positives were minimized.
Supplementary Figure 4 Fragment ions specific to biotin phenol can be used to enhance spectral interpretation.
(A) Structure of biotinylated tyrosine (B) Structure and mass of the immonium ion of biotinylated tyrosine (C) Structure and mass of the biotin phenol cleavage product produced upon HCD fragmentation.
Supplementary Figure 5 Majority of biotinylated peptides identified in antibody enrichment experiments are modified at tyrosine.
Bar plot shows the number of distinct biotinylation sites identified at tyrosine (Y), tryptophan (W), and cysteine (C) from SILAC labeled HEK 293T cells stably expressing matrix-APEX2. The percent of each site at a given amino acid is calculated relative to the total number of biotinylation sites.
(Left Panel) Many inner membrane proteins are imported via the Stop-Transfer pathway, where proteins are arrested at the TIM23 complex and laterally instead into the membrane. (Right Panel) Another, smaller class of inner membrane proteins are processed via a Conservative Sorting pathway. In this pathway, proteins initially translocate to the matrix prior to insertion in the inner membrane. We propose that biotinylation of Y141 may occur on a processing intermediate of LETM1 if the protein is processed via the Conservative Sorting pathway.21Neupert, W. & Herrmann, J.M. Translocation of proteins into mitochondria. Annu Rev Biochem 76, 723-749 (2007).
Supplementary Figures 1–6 and Supplementary Tables 4 and 9 (PDF 1123 kb)
NHS-Biotin Peptide Spike-ins (XLSX 59612 kb)
Comparison of anti-biotin antibody and NeutrAvidin enrichment (XLSX 6946 kb)
Proteins: APEX labelling Streptavidin Enrichment (XLSX 189 kb)
Biotinylation Sites: APEX labelling Streptavidin Enrichment (XLSX 21 kb)
Biotinylation Sites: APEX labelling Anti-Biotin Antibody Enrichment; all (XLSX 625 kb)
Biotinylation Sites: APEX labelling Anti-Biotin Antibody Enrichment; >= 2 replicates (XLSX 373 kb)
Coverage of OXPHOS proteins (XLSX 12 kb)
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Udeshi, N., Pedram, K., Svinkina, T. et al. Antibodies to biotin enable large-scale detection of biotinylation sites on proteins. Nat Methods 14, 1167–1170 (2017). https://doi.org/10.1038/nmeth.4465
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