Abstract

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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Acknowledgements

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.

Author information

Affiliations

  1. Proteomics, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA.

    • Namrata D Udeshi
    • , Tanya Svinkina
    • , Shaunt Fereshetian
    • , Samuel A Myers
    • , Ozan Aygun
    • , Karsten Krug
    • , Karl Clauser
    • , Tslil Ast
    • , Vamsi K Mootha
    •  & Steven A Carr
  2. Departments of Genetics, Biology, and Chemistry, Stanford University, Stanford, California, USA.

    • Kayvon Pedram
    •  & Alice Y Ting
  3. DRI2 LLC, Littleton, Massachusetts, USA.

    • Dominic Ryan
  4. Howard Hughes Medical Institute, Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA.

    • Tslil Ast
    •  & Vamsi K Mootha

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Contributions

N.D.U. and S.A.C. conceived of the study; N.D.U., K.P., T.S., and S.F. performed experiments; N.D.U., K.P., T.S., S.F., S.A.M., O.A., K.K., K.C., D.R., T.A., V.K.M., A.Y.T., and S.A.C. contributed to experimental design, data analysis, and data interpretation. N.D.U. and S.A.C. wrote the manuscript with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Namrata D Udeshi or Steven A Carr.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–6 and Supplementary Tables 4 and 9

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Protocol

Excel files

  1. 1.

    Supplementary Table 1

    NHS-Biotin Peptide Spike-ins

  2. 2.

    Supplementary Table 2

    Comparison of anti-biotin antibody and NeutrAvidin enrichment

  3. 3.

    Supplementary Table 3

    Proteins: APEX labelling Streptavidin Enrichment

  4. 4.

    Supplementary Table 5

    Biotinylation Sites: APEX labelling Streptavidin Enrichment

  5. 5.

    Supplementary Table 6

    Biotinylation Sites: APEX labelling Anti-Biotin Antibody Enrichment; all

  6. 6.

    Supplementary Table 7

    Biotinylation Sites: APEX labelling Anti-Biotin Antibody Enrichment; >= 2 replicates

  7. 7.

    Supplementary Table 8

    Coverage of OXPHOS proteins

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DOI

https://doi.org/10.1038/nmeth.4465