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Engineered SH2 domains with tailored specificities and enhanced affinities for phosphoproteome analysis

Protein phosphorylation is the most abundant post‐translational modification in cells. Src homology 2 (SH2) domains specifically recognize phosphorylated tyrosine (pTyr) residues to mediate signaling cascades. A conserved pocket in the SH2 domain binds the pTyr side chain and the EF and BG loops determine binding specificity. By using large phage‐displayed libraries, we engineered the EF and BG loops of the Fyn SH2 domain to alter specificity. Engineered SH2 variants exhibited distinct specificity profiles and were able to bind pTyr sites on the epidermal growth factor receptor, which were not recognized by the wild‐type Fyn SH2 domain. Furthermore, mass spectrometry showed that SH2 variants with additional mutations in the pTyr‐binding pocket that enhanced affinity were highly effective for enrichment of diverse pTyr peptides within the human proteome. These results showed that engineering of the EF and BG loops could be used to tailor SH2 domain specificity, and SH2 variants with diverse specificities and high affinities for pTyr residues enabled more comprehensive analysis of the human phosphoproteome.