Clickable Covalent Ligand Profiling for Proteome-Wide Target Quantification
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Solution Overview
Problem
Current methods are inadequate for identifying and quantifying the binding interactions of reactive chemical probes with various components in whole cell proteomes.
Innovation Solution
A method involving electrophilic compounds with clickable tags, such as alkyne moieties, to covalently bind to nucleophilic amino acids in proteins, followed by mass spectrometry analysis to identify and quantify protein targets, using isobaric mass tags for comparative analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional binding interaction methods are used, then identification of protein targets is possible, but the ability to simultaneously identify and quantify interactions with thousands of proteins is insufficient
Solution Approach 1:
The method segments the proteome analysis by using competitive binding assays with specific competitor compounds that target different protein families. This allows parallel identification and quantification of interactions with multiple protein targets simultaneously, achieving high throughput while maintaining measurement precision through controlled competition
Solution Approach 2:
The patent introduces clickable tags (alkyne or azide moieties) as intermediaries that covalently bind to nucleophilic amino acids in protein targets. These tags serve as handles for subsequent detection and quantification via mass spectrometry, enabling simultaneous identification and measurement of binding interactions across thousands of proteins
2Reliability
If reactive chemical probes are used to identify protein targets, then covalent binding can be detected, but the complexity of analyzing interactions in whole cell proteomes increases
Solution Approach 1:
The method extracts the clickable tag portion from the probe compound, which serves as a detectable handle while the electrophilic moiety performs the covalent binding function. This separation allows the probe to reliably detect covalent interactions while simplifying the analysis by focusing only on the tag-containing protein complexes
Solution Approach 2:
The patent performs preliminary covalent binding of the electrophilic moiety to nucleophilic amino acids in protein targets before detection. This preliminary action establishes stable covalent adducts that can be reliably detected and quantified, simplifying the overall analysis complexity while maintaining high reliability
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
Enables the simultaneous identification and quantification of covalent interactions with thousands of proteins, facilitating the development of potential therapeutic compounds.
Implementation Method 1
The predominant form of such covalent modification occurs between an electrophilic moiety in the drug or drug candidate and a nucleophilic amino acid in the protein
Implementation Method 2
the clickable tag is covalently linked to a detectable label
Data Source
AI summary
The present disclosure provides a profiling method based on comparative mass spectrometry analysis for identifying and quantifying the covalent interactions of electrophilic compounds with diverse proteins in complex proteomes, as well as compositions for performing the method.