Engineered SH2 Domains for Targeted Phosphoproteomics

ACS Chem Biol. 2022 Jun 17;17(6):1472-1484. doi: 10.1021/acschembio.2c00051. Epub 2022 May 25.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, N.I.H., Extramural

MeSH terms

  • Humans
  • Mass Spectrometry
  • Phosphotyrosine / analysis
  • Phosphotyrosine / chemistry
  • Phosphotyrosine / metabolism
  • Protein Binding
  • Proteome* / metabolism
  • src Homology Domains*

Substances

  • Proteome
  • Phosphotyrosine