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.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $20.83 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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).
Integrated supplementary information
Oligonucleotide sequences encoding human phosphoserine phosphosite library
Mass spectrometry analysis of mode #1 phosphosite library preparations
Phospho-specific antibodies for evaluation of recombinant phosphosites
Phosphosites observed by Hi-P using 14-3-3β and SepOTSλ
Phosphosites observed by Hi-P using 14-3-3σ and SepOTSλ
Positional amino acid frequencies in pSer-encoding phosphosite populations interacting with 14-3-3β by Hi-P
Positional amino acid frequencies in pSer-encoding phosphosite populations interacting with 14-3-3σ by Hi-P
Full-length recombinant human phosphoprotein genes identified as candidate 14-3-3β candidate interactors, synthesized for pull-down studies
Phosphosites observed by Hi-P using NEDD4 WW2 and SepOTSλ
Phosphosites observed by Hi-P using NEDD4 WW2 and supD tRNA
Phosphosites observed by Hi-P using NEDD4-2 WW2 and SepOTSλ
Phosphosites observed by Hi-P using NEDD4-2 WW2 and supD tRNA
Co-immunoprecipitation mass spectrometry data analysis
HTS sequencing from biological triplicate Hi-P experiments
Orthogonal primer sequences for phosphosite DNA library amplification
Synthesized DNA sequences