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. Author manuscript; available in PMC: 2019 Jun 24.
Published in final edited form as: Nat Biotechnol. 2018 Jun 11;36(7):638–644. doi: 10.1038/nbt.4150

Figure 2: Expression and LC-MS/MS validation of the synthetic human serine phosphoproteome.

Figure 2:

(a) Mode #1 phosphosite library configuration for overexpression in E. coli. The mode #1 plasmid library is introduced into C321.ΔA with either SepOTSλ or supD tRNA to express pSer- or Ser-containing mode #1 phosphosite libraries, respectively. (b) The plasmid library used for mode #1 phosphosite expression encodes ~94% of the 110,139 designed recombinant phosphosites as determined by HTS analysis. (c) Western blot from Phos-tag acrylamide gel illustrates broad mobility shift of recombinant pSer-encoding phosphosite library, either with the GST fusion tag or after enzymatic removal of the GST tag. Similar results were obtained for two other independently purified mode #1 protein libraries. (d) >36,000 unique phosphopeptides generated using SepOTSλ containing pSer at the encoded position were directly observed by LC-MS/MS, and evidence for >56,000 unique phosphosite library members was observed in total across all sample preparations (mode #1 phosphosite libraries made with either SepOTSλ or supD tRNA).