Engineering human SH2 domains for targeted phospho-proteomics

A comprehensive analysis of the phosphoproteome is essential for understanding molecular mechanisms of human diseases. However, current tools to enrich phosphotyrosine are limited in their applicability and scope. Here, we engineered new superbinder SH2 domains that enrich diverse sets of phosphotyrosine peptides. We used phage display to select a Fes SH2 domain variant with high affinity for phosphotyrosine (superFes-SH2, sFes1) and solved the structure of sFes1 bound to a phosphopeptide. We performed systematic structure-function analyses of the superbinding mechanisms of sFes1 and superSrc-SH2 (sSrc1), another SH2-superbinder. We grafted the superbinder motifs from sFes1 and sSrc1 into 17 additional SH2 domains and confirmed increased binding affinity for specific phosphopeptides. Using mass spectrometry, we demonstrated that SH2 superbinders have distinct specificity profiles and superior capabilities to enrich phosphotyrosine peptides. Finally, combinations of SH2 superbinders as affinity purification tools showed that unique subsets of phosphopeptides can be enriched with unparalleled depth and coverage.