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Quantifying protein–protein interactions in high throughput using protein domain microarrays

Nature Protocols volume 5, pages 773–790 (2010)Cite this article

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Abstract

Protein microarrays provide an efficient way to identify and quantify protein–protein interactions in high throughput. One drawback of this technique is that proteins show a broad range of physicochemical properties and are often difficult to produce recombinantly. To circumvent these problems, we have focused on families of protein interaction domains. Here we provide protocols for constructing microarrays of protein interaction domains in individual wells of 96-well microtiter plates, and for quantifying domain–peptide interactions in high throughput using fluorescently labeled synthetic peptides. As specific examples, we will describe the construction of microarrays of virtually every human Src homology 2 (SH2) and phosphotyrosine binding (PTB) domain, as well as microarrays of mouse PDZ domains, all produced recombinantly in Escherichia coli. For domains that mediate high-affinity interactions, such as SH2 and PTB domains, equilibrium dissociation constants (KDs) for their peptide ligands can be measured directly on arrays by obtaining saturation binding curves. For weaker binding domains, such as PDZ domains, arrays are best used to identify candidate interactions, which are then retested and quantified by fluorescence polarization. Overall, protein domain microarrays provide the ability to rapidly identify and quantify protein–ligand interactions with minimal sample consumption. Because entire domain families can be interrogated simultaneously, they provide a powerful way to assess binding selectivity on a proteome-wide scale and provide an unbiased perspective on the connectivity of protein–protein interaction networks.

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Figure 1: Protein domain microarrays.
Figure 2: Quantifying domain–peptide interactions in high throughput using protein domain microarrays.
Figure 3: Using protein domain microarrays to identify and quantify domain–peptide binding interactions.
Figure 4: Microarrays in microtiter plates.

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Acknowledgements

We thank J.-R. Chen, G. Koytiger and M. Sevecka for helpful contributions to these protocols, particularly in regards to data processing and analysis. This work was supported by awards from the W.M. Keck Foundation, the Arnold and Mabel Beckman Foundation, and the Camille and Henry Dreyfus Foundation, and by grants from the National Institutes of Health (1 RO1 GM072872 and 1 R33 CA128726). A.K. was supported in part by the National Institutes of Health Molecular, Cellular and Chemical Biology Training grant (5 T32 GM07598) and A.G. was the recipient of an NSF Graduate Research Fellowship.

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Author notes

  1. Andrew Gordus & Michael A Stiffler

    Present address: Present addresses: The Rockefeller University, New York, New York, USA (A.G.); UT Southwestern Medical Center, Dallas, Texas, USA (M.A.S.),

  2. Alexis Kaushansky and John E Allen: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA

    Alexis Kaushansky, John E Allen, Andrew Gordus, Michael A Stiffler, Ethan S Karp, Bryan H Chang & Gavin MacBeath

  2. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA

    Alexis Kaushansky

  3. Program in Biophysics, Harvard University, Cambridge, Massachusetts, USA

    Andrew Gordus

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Contributions

A.K., J.E.A. and G.M. wrote the article. A.G. developed initial protocols for printing and probing microarrays of SH2 and PTB domains. A.K. contributed to the further development of these protocols. M.A.S. developed initial protocols for quantifying PDZ domain–peptide interactions using fluorescence polarization. J.E.A. developed peptide synthesis protocols and contributed to the development of FP assays. E.S.K. assisted in the development of FP assays and to the processing of SH2/PTB domain microarray data. B.H.C. developed peptide purification protocols and contributed to the development of FP protocols.

Corresponding author

Correspondence to Gavin MacBeath.

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Competing interests

Gavin MacBeath is an advisor for and stockholder in Merrimack Pharmaceuticals, Inc., Makoto Life Sciences, Inc., and Aushon BioSystems, Inc.

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Kaushansky, A., Allen, J., Gordus, A. et al. Quantifying protein–protein interactions in high throughput using protein domain microarrays. Nat Protoc 5, 773–790 (2010). https://doi.org/10.1038/nprot.2010.36

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