Method for proteome-wide discovery of covalent ligands and compositions thereof
Inventors
Cao, Ping • Yuan, Yuan • Zhang, Chao
Assignees
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Publication Number
US-12392778-B2
Publication Date
2025-08-19
Expiration Date
Abstract
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.
Core Innovation
The invention relates to a method for identifying a protein target using a proteome-wide comparative mass spectrometry approach. A first protein-containing sample comprising the protein target is contacted with a competitor compound comprising a core structure and an electrophilic moiety, where the electrophilic moiety binds a nucleophilic amino acid residue in the protein target, and the competitor compound does not comprise a terminal clickable tag.
A second contacting step labels the protein targets with a probe compound that includes a core structure and an electrophilic moiety identical to those of the competitor compound, and further includes a terminal clickable tag. The terminal clickable tag is specified as a terminal alkyne moiety, enabling detection of probe-labeled proteins by identifying probe compound-modified proteins in the competitor-treated first sample compared to those in a second protein-containing sample not contacted with the competitor.
The method is supported by claim-dependent embodiments that include derivatizing probe compound-modified proteins by attaching a detectable label through a clickable tag. Additional refinements specify selectable nucleophilic amino acid residues in the protein target, protease digestion of an enriched protein target, and comparative analysis using LC-MS/MS with a tandem mass tag (TMT), including embodiments that quantify binding interactions between the competitor compound and the protein target.
Claims Coverage
The independent claim defines a competitor-versus-probe labeling workflow with terminal alkyne click functionality and comparative detection, totaling three main inventive concepts. Dependent claims add constraints and analytical refinements, including detectable labeling, selectable nucleophilic residues, proteome splitting into equivalent samples, protease digestion, and optional quantification using LC-MS/MS with TMT.
Competitor compound lacking a terminal clickable tag with identical core and electrophile binding
Contacting a first protein-containing sample comprising a protein target with a competitor compound comprising a core structure and an electrophilic moiety to obtain a sample treated with the competitor compound, wherein the electrophilic moiety binds a nucleophilic amino acid residue in the protein target, and wherein the competitor compound does not comprise a terminal clickable tag.
Probe compound with identical core and electrophile and terminal alkyne clickable tag
Contacting the sample treated with the competitor compound with a probe compound comprising a core structure and an electrophilic moiety identical to the core structure and the electrophilic moiety of the competitor compound, and a terminal clickable tag, wherein the clickable tag is a terminal alkyne moiety.
Comparative detection between competitor-treated sample and competitor-naive sample
Contacting a second protein-containing sample comprising the protein target with the probe compound, wherein the second protein-containing sample has not been contacted with the competitor compound, and identifying the protein target by detecting the probe compound-modified proteins in the first protein-containing sample as compared to those in the second protein-containing sample.
Overall claim coverage centers on identifying a covalently engaged protein target by competitor pre-blocking without a terminal clickable tag, followed by probe labeling using an identical electrophilic core plus a terminal alkyne clickable tag, with target identification by comparative detection of probe-modified proteins between competitor-treated and competitor-naive samples.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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