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Discovery of protein interactions using parallel analysis of translated ORFs (PLATO)

Nature Protocols 9, 90–103 (2014) | Download Citation

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Abstract

Parallel analysis of translated open reading frames (ORFs) (PLATO) can be used for the unbiased discovery of interactions between full-length proteins encoded by a library of 'prey' ORFs and surface-immobilized 'bait' antibodies, polypeptides or small-molecular-weight compounds. PLATO uses ribosome display (RD) to link ORF-derived mRNA molecules to the proteins they encode, and recovered mRNA from affinity enrichment is subjected to analysis using massively parallel DNA sequencing. Compared with alternative in vitro methods, PLATO provides several advantages including library size and cost. A unique advantage of PLATO is that an alternative reverse transcription–quantitative PCR (RT-qPCR) protocol can be used to test binding of specific, individual proteins. To illustrate a typical experimental workflow, we demonstrate PLATO for the identification of the immune target of serum antibodies from patients with inclusion body myositis (IBM). Beginning with an ORFeome library in an RD vector, the protocol can produce samples for deep sequencing or RT-qPCR within 4 d.

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Acknowledgements

IBM patient sera were kindly provided by Stephen A. Greenberg (Brigham and Women's Hospital). The ORFeome library v5.1 was provided by the Center for Cancer Systems Biology (CCSB) at the Dana-Farber Cancer Institute (Boston, Massachusetts). This work was supported in part by a grant from the Leona M. and Harry B. Helmsley Charitable Trust. S.J.E. is an investigator with the Howard Hughes Medical Institute.

Author information

    • H Benjamin Larman
    •  & Jian Zhu

    Present addresses: The Scripps Research Institute, La Jolla, California, USA (H.B.L.); University of Rochester Medical Center, Rochester, New York, USA (J.Z.).

Affiliations

  1. Harvard-MIT Division of Health Sciences and Technology, Cambridge, Massachusetts, USA.

    • H Benjamin Larman

  2. Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • H Benjamin Larman

  3. Department of Genetics, Harvard University Medical School, Boston, Massachusetts, USA.

    • H Benjamin Larman
    • , Stephen J Elledge
    •  & Jian Zhu

  4. Department of Medicine, Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts, USA.

    • H Benjamin Larman
    • , Anthony C Liang
    • , Stephen J Elledge
    •  & Jian Zhu

  5. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.

    • H Benjamin Larman
    • , Anthony C Liang
    • , Stephen J Elledge
    •  & Jian Zhu

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Contributions

H.B.L., S.J.E. and J.Z. developed the protocol. H.B.L. and J.Z. wrote the paper. J.Z. performed the PLATO screen with IBM samples. A.C.L. performed the immunofluorescence.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Stephen J Elledge or Jian Zhu.

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DOI

https://doi.org/10.1038/nprot.2013.167

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