Phosphopeptide-encoding oligonucleotide libraries for site-specific phosphoprotein detection

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Solution Overview

Problem

Current methods are limited in comprehensively understanding and studying protein phosphorylation due to the inability to generate phosphoproteins with the specificity of natural systems, hindering the identification of kinases responsible for phosphorylation events and the functional significance of phosphorylation sites in protein-protein interactions.

Innovation Solution

A library of phosphopeptide-encoding oligonucleotides is synthesized to encode phosphoserine residues and their surrounding sequences, allowing for the site-specific incorporation of phosphoserine into recombinant proteins, enabling the detection of phosphorylation-dependent protein-protein interactions using genetically encoded systems.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If genetically encoded nonstandard amino acids are used to incorporate phosphoserine into proteins, then site-specific phosphorylation can be achieved, but the technology is limited to studying only a few proteins

Engineering Contradiction:

Improvesite-specific phosphorylation detectionVSAvoidnumber of proteins that can be studied

Core Design Contradiction:

Measurement precisionVSAdaptability or versatility

Solution Approach 1:

The invention creates a universal phosphoprotein library system that can be applied to study any phosphoprotein in the human phosphoproteome. By developing a comprehensive library containing thousands of phosphoprotein variants with site-specific phosphoserine incorporation, the system moves beyond studying only a few proteins to enabling broad-scale analysis of phosphorylation-dependent interactions across the entire phosphoproteome.

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Reliability

If heterogeneous and low-stoichiometric phosphorylation is present in cells, then natural phosphorylation states are maintained, but the relevance of phosphorylation in assembled complexes is difficult to ascertain

Engineering Contradiction:

Improvenatural phosphorylation stateVSAvoidphosphorylation-dependent interactions

Core Design Contradiction:

ReliabilityVSDifficulty of detecting and measuring

Solution Approach 1:

The invention changes the stoichiometry parameter of phosphorylation by using genetically encoded site-specific phosphoserine incorporation to create homogeneous, high-stoichiometric phosphoprotein libraries. This allows phosphorylation to be present at near-100% occupancy at specific sites, transforming the natural heterogeneous/low-stoichiometric state into a controlled homogeneous/high-stoichiometric state that enables reliable detection of phosphorylation-dependent interactions.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention creates recombinant copies of phosphoproteins with defined phosphorylation states that mirror natural phosphorylation sites. By synthesizing library members that replicate specific phosphorylation events with high fidelity and uniformity, the system produces copy versions of phosphoproteins that are easier to detect and analyze while maintaining the biological relevance of the phosphorylation sites.

Inventive Principle:

Principle #26Copying

3Loss of information

If comprehensive phosphoproteome analysis is performed, then functional landscape of phosphorylation can be revealed, but the complexity of identifying kinase-substrate connections increases

Engineering Contradiction:

Improvephosphorylation cascade understandingVSAvoidkinase-substrate connection identification

Core Design Contradiction:

Loss of informationVSDevice complexity

Solution Approach 1:

The invention segments the complex problem of kinase-substrate identification by creating a library of individual phosphoprotein variants, each representing a specific phosphorylation site. By analyzing interactions of individual library members rather than attempting to analyze the entire phosphoproteome simultaneously, the system breaks down the complex identification problem into manageable segments that can be studied systematically.

Inventive Principle:

Principle #1Segmentation

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 the identification of thousands of recombinant phosphoproteins by mass spectrometry and reveals novel candidate interactions, providing a comprehensive view of the human phosphoproteome and its functional landscape, with a 30-fold enrichment of known interactions and discovery of over 600 novel candidates.

Implementation Method 1

Methodology Applied

Scientific EffectGenetic encoding:

Implementation Method 2

This approach enables the identification of thousands of recombinant phosphoproteins by mass spectrometry and reveals novel candidate interactions

Methodology Applied

Scientific EffectMass spectrometry:

Data Source

PatentUS11718849B2Phosphopeptide-encoding oligonucleotide libraries and methods for detecting phosphorylation-dependent molecular interactions

Publication Date: 2023.08.08 AGILENT TECHNOLOGIES INC

AI summary

The present invention relates to libraries of phosphopeptide-encoding oligonucleotides and methods of preparing such libraries. The present invention also relates to methods of detecting, visualizing, or screening for phosphorylation-dependent protein-protein interactions using recombinant phosphopeptides and/or phosphopeptide-encoding oligonucleotides. The present invention also relates to sets or kits of oligonucleotides having regions that encode phosphopeptides.