Variant SH2 Superbinders for Phosphotyrosine Peptide Profiling
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Solution Overview
Problem
Current methods for detecting protein tyrosine phosphorylation, particularly in the context of protein kinases and immune function, are limited in their ability to comprehensively identify and quantify tyrosine phosphorylation sites, especially in biological samples, due to low affinity of traditional SH2 domains for pTyr residues, leading to incomplete coverage of the Tyr phosphoproteome.
Innovation Solution
The use of variant SH2 domains, referred to as Superbinders, with enhanced affinity for pTyr residues, which are combined with mass spectrometry techniques for enrichment and identification of tyrosine-phosphorylated peptides, allowing for the detection of hundreds of Tyr phosphosites and quantification of their phosphorylation status in minute samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional SH2 domains are used for detecting tyrosine phosphorylation, then the method is simple and well-established, but the affinity for pTyr residues is low resulting in incomplete coverage of the Tyr phosphoproteome
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of SH2 domains to create variant SH2 domains with enhanced binding affinity for phosphotyrosine residues. Specifically, mutations are introduced in the pTyr-binding pocket to optimize interactions with pTyr-containing peptides, thereby improving the reliability and coverage of tyrosine phosphorylation detection without fundamentally changing the overall detection methodology.
2Measurement precision
If variant SH2 domains with enhanced affinity are used, then the sensitivity and coverage of phosphotyrosine detection is improved, but the complexity of the system increases
Solution Approach 1:
The patent modifies binding parameters of SH2 domains by introducing specific amino acid substitutions that enhance affinity for phosphotyrosine residues. These parameter changes in the protein structure directly improve measurement precision and sensitivity of phosphotyrosine detection, allowing for more accurate quantification of tyrosine phosphorylation sites.
Solution Approach 2:
The patent creates multiple copies of variant SH2 domains with different specificities to detect various phosphotyrosine motifs. By engineering a family of SH2 domain variants, each with optimized binding characteristics for different peptide sequences, the system achieves comprehensive coverage of the phosphoproteome while maintaining manageable complexity through modular design.
3Loss of information
If comprehensive profiling of tyrosine phosphorylation is achieved, then detailed insights into kinase activity and immune signaling are obtained, but the amount of sample and resources required increases
Solution Approach 1:
The enhanced binding affinity of variant SH2 domains for phosphotyrosine residues enables more efficient enrichment of phosphopeptides from biological samples. This parameter improvement in binding strength allows for comprehensive profiling of tyrosine phosphorylation with reduced sample requirements, as the high-affinity interactions enable effective capture and detection of phosphopeptides even in limited quantities.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach enables systematic profiling of protein tyrosine phosphorylation, providing detailed insights into kinase activity and immune signaling, including disease states and treatment responses, with improved sensitivity and comprehensive coverage compared to traditional methods.
Implementation Method 1
contacting the test sample with an SH2 Superbinder in order to bind pTyr-including peptides contained in the test sample with the SH2 Superbinder
Implementation Method 2
identifying the bound pTyr-including peptides
Data Source
AI summary
There is provided method of profiling protein tyrosine phosphorylation of a sample, the method comprising: contacting the sample with an SH2 Superbinder in order to bind pTyr-including peptides contained in the sample with the SH2 Superbinder; isolating the bound pTyr-including peptides from the sample; and identifying the isolated pTyr-including peptides.