Engineered SH2 Domains for Targeted Phosphoproteomics.

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Understanding the phosphoproteome is crucial for deciphering human disease mechanisms.
• Current methods for enriching phosphotyrosine (pTyr) peptides have limitations in scope and applicability.

Purpose of the Study:

• To engineer novel Src-Homology 2 (SH2) superbinder domains with enhanced affinity and specificity for phosphotyrosine (pTyr) peptides.
• To improve the enrichment and analysis of the phosphoproteome for disease research.

Main Methods:

• Phage display was used to select and engineer superbinder SH2 domains (e.g., superFes, sFes¹).
• Structural and functional analyses were performed on engineered SH2 superbinder variants.
• Mass spectrometry (MS) and affinity purification (AP) techniques were employed to assess enrichment capabilities.

Main Results:

• Engineered SH2 superbinders (sFes¹, sSrc¹) demonstrated high affinity for pTyr-peptides.
• Grafting superbinder motifs into other SH2 domains confirmed increased binding affinity.
• SH2 superbinders exhibited distinct specificity profiles and superior pTyr-peptide enrichment capabilities via MS.
• Combinations of SH2 superbinders enabled deep and broad enrichment of unique pTyr-peptide subsets.

Conclusions:

• Novel SH2 superbinders significantly enhance the enrichment of diverse phosphotyrosine-containing peptides.
• These engineered tools offer improved depth and coverage for phosphoproteome analysis.
• The developed SH2 superbinders represent a valuable advancement for studying disease mechanisms at the molecular level.