R2HaPpY: Rapid-robust phosphotyrosine peptide enrichment using HaloTag-Src SH2 pY superbinder

Area of Science:

• Molecular Biology
• Cell Signaling
• Biochemistry

Background:

• Phosphotyrosine signaling is crucial for cellular functions like proliferation and immune response.
• Analyzing phosphotyrosine signaling comprehensively is challenging due to expensive reagents and complex protocols.
• Previous automated methods improved sample preparation but reagent preparation remained a bottleneck.

Purpose of the Study:

• To optimize an automated phosphotyrosine enrichment method for faster and more cost-effective reagent preparation.
• To enhance the efficiency of phosphotyrosine peptide binding for improved detection sensitivity.
• To enable a more comprehensive and quantitative analysis of tyrosine phosphorylation dynamics.

Main Methods:

• Fused an SH2 phosphotyrosine superbinder to the HaloTag protein for reagent development.
• Developed a method for cost-effective preparation of enrichment beads directly from bacterial lysate.
• Applied the optimized method to analyze phosphotyrosine sites in EGF-stimulated HeLa cells.

Main Results:

• Expedited reagent preparation from days to hours.
• Achieved higher binding efficiency for phosphotyrosine peptides compared to existing reagents.
• Detected and quantified 1,651 unique phosphotyrosine sites, including 878 regulated sites, with ~1 mg of input peptides.

Conclusions:

• The streamlined and sensitive method significantly improves the efficiency of phosphotyrosine enrichment.
• This approach facilitates comprehensive mapping of tyrosine phosphorylation dynamics.
• Enables broader integration of phosphotyrosine signaling data into multiomic and network models for diverse biological studies.