Post-translational phosphorylation is essential to human cellular processes, but the transient, heterogeneous nature of this modification complicates its study in native systems1,2,3. We developed an approach to interrogate phosphorylation and its role in protein-protein interactions on a proteome-wide scale. We genetically encoded phosphoserine in recoded E. coli4,5,6 and generated a peptide-based heterologous representation of the human serine phosphoproteome. We designed a single-plasmid library encoding >100,000 human phosphopeptides and confirmed the site-specific incorporation of phosphoserine in >36,000 of these peptides. We then integrated our phosphopeptide library into an approach known as Hi-P to enable proteome-level screens for serine-phosphorylation-dependent human protein interactions. Using Hi-P, we found hundreds of known and potentially new phosphoserine-dependent interactors with 14-3-3 proteins and WW domains. These phosphosites retained important binding characteristics of the native human phosphoproteome, as determined by motif analysis and pull-downs using full-length phosphoproteins. This technology can be used to interrogate user-defined phosphoproteomes in any organism, tissue, or disease of interest.

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We thank J. Niemann for his assistance with selecting phospho-specific antibodies to probe phosphosite synthesis, P. Anderson for assistance with oligonucleotide library synthesis, the Yale Center for Genome Analysis for high-throughput sequencing and K. Mohler for helpful comments on the manuscript. K.W.B. is supported by the National Science Foundation Graduate Research Fellowship under grant no. DGE-1122492. R.S. received support from the Raymond and Beverly Sackler Institute for Biological, Physical and Engineering Sciences at Yale University and from Southern Connecticut State University School of Graduate Studies, Research and Innovation. J.R. is supported by the US National Institutes of Health (NIH; GM117230, GM125951, DK0174334, CA209992). F.J.I. acknowledges support from the NIH (GM117230, GM125951, CA209992), DARPA (HR0011-15-C-0091), NSF (MCB-1714860, CHE-1740549), DuPont, and the Arnold and Mabel Beckman Foundation. M.G. is supported by the NIH (HG008126).

Author information


  1. Department of Cellular & Molecular Physiology, Yale University, New Haven, Connecticut, USA.

    • Karl W Barber
    • , Svetlana Rogulina
    •  & Jesse Rinehart
  2. Systems Biology Institute, Yale University, West Haven, Connecticut, USA.

    • Karl W Barber
    • , Paul Muir
    • , Richard V Szeligowski
    • , Svetlana Rogulina
    • , Mark Gerstein
    • , Farren J Isaacs
    •  & Jesse Rinehart
  3. Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA.

    • Paul Muir
    •  & Farren J Isaacs
  4. Biology Department, Southern Connecticut State University, New Haven, Connecticut, USA.

    • Richard V Szeligowski
  5. Program in Computational Biology and Bioinformatics, Yale University, New Haven, Connecticut, USA.

    • Mark Gerstein
  6. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.

    • Mark Gerstein
  7. Department of Computer Science, Yale University, New Haven, Connecticut, USA.

    • Mark Gerstein
  8. Agilent Laboratories, Agilent Technologies, Santa Clara, California, USA.

    • Jeffrey R Sampson


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J.R., F.J.I. and K.W.B. conceived the project, designed the experiments and analyzed the data. K.W.B, R.V.S. and S.R. performed molecular cloning, protein expression/purification and western blotting. K.W.B. performed mass spectrometry and Hi-P sample preparation, data collection, and analysis. K.W.B., P.M., M.G. and F.J.I. performed high-throughput sequencing and network analysis. K.W.B and F.J.I. developed oligonucleotide design and library preparation strategies. J.R.S. provided guidance on DNA construct design strategies using Agilent's Oligo Library Synthesis. J.R. and K.W.B. wrote the manuscript with input from all of the other authors.

Competing interests

J.R., F.J.I., K.W.B. and J.R.S. have filed a provisional patent application with the US Patent and Trademark Office (US Patent Application No. 62/639,279) related to this work.

Corresponding author

Correspondence to Jesse Rinehart.

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Supplementary information

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    Supplementary Text and Figures

    Supplementary Figures 1–12

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    Life Sciences Reporting Summary

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    Supplementary Notes

    Supplementary Notes 1–11

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    Supplementary Data 1

    Oligonucleotide sequences encoding human phosphoserine phosphosite library

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    Supplementary Data 2

    Mass spectrometry analysis of mode #1 phosphosite library preparations

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    Supplementary Data 3

    Phospho-specific antibodies for evaluation of recombinant phosphosites

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    Supplementary Data 4

    Phosphosites observed by Hi-P using 14-3-3β and SepOTSλ

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    Supplementary Data 5

    Phosphosites observed by Hi-P using 14-3-3σ and SepOTSλ

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    Supplementary Data 6

    Positional amino acid frequencies in pSer-encoding phosphosite populations interacting with 14-3-3β by Hi-P

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    Supplementary Data 7

    Positional amino acid frequencies in pSer-encoding phosphosite populations interacting with 14-3-3σ by Hi-P

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    Supplementary Data 8

    Full-length recombinant human phosphoprotein genes identified as candidate 14-3-3β candidate interactors, synthesized for pull-down studies

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    Supplementary Data 9

    Phosphosites observed by Hi-P using NEDD4 WW2 and SepOTSλ

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    Supplementary Data 10

    Phosphosites observed by Hi-P using NEDD4 WW2 and supD tRNA

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    Supplementary Data 11

    Phosphosites observed by Hi-P using NEDD4-2 WW2 and SepOTSλ

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    Supplementary Data 12

    Phosphosites observed by Hi-P using NEDD4-2 WW2 and supD tRNA

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    Supplementary Data 13

    Co-immunoprecipitation mass spectrometry data analysis

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    Supplementary Data 14

    HTS sequencing from biological triplicate Hi-P experiments

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    Supplementary Data 15

    Orthogonal primer sequences for phosphosite DNA library amplification

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    Supplementary Data 16

    Synthesized DNA sequences

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    Supplementary Data 17

    Primer sequences

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