Engineered SH2 Domains for Targeted Phosphoproteomics

A comprehensive
analysis of the phosphoproteome is essential for
understanding molecular mechanisms of human diseases. However, current
tools used to enrich phosphotyrosine (pTyr) are limited in their applicability
and scope. Here, we engineered new superbinder Src-Homology 2 (SH2)
domains that enrich diverse sets of pTyr-peptides. We used phage display
to select a Fes-SH2 domain variant (superFes; sFes1) with
high affinity for pTyr and solved its structure bound to a pTyr-peptide.
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 pTyr-peptides.
Using mass spectrometry (MS), we demonstrated that SH2 superbinders
have distinct specificity profiles and superior capabilities to enrich
pTyr-peptides. Finally, using combinations of SH2 superbinders as
affinity purification (AP) tools we showed that unique subsets of
pTyr-peptides can be enriched with unparalleled depth and coverage.