Methods for discovering protein-protein interactions
Inventors
Pierobon, Mariaelena • Petricoin, Emanuel • Baldelli, Elisa
Assignees
Publication Number
US-11408896-B2
Publication Date
2022-08-09
Expiration Date
2039-06-19
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Abstract
An embodiment relates to a method, comprising: obtaining a biological sample, performing a protein complex immunoprecipitation (Co-IP) on the sample, performing a reverse phase protein array (RPPA) on the sample after performing the protein complex immunoprecipitation, and identifying one or more protein complexes. A further embodiment relates to a method of treatment, comprising: obtaining a tissue sample from a patient, performing Co-IP then RPPA on the sample, identifying a protein complex, determining whether the protein complex comprises known protein drug targets, and treating the patient with a drug that interacts with the known protein drug targets.
Core Innovation
The invention describes a method for identifying protein-protein interactions (PPI) and protein complexes in any cell type. The method consists of obtaining a biological sample, performing co-immunoprecipitation (Co-IP) to isolate protein complexes, then using reverse phase protein array (RPPA) for multiplexed identification and characterization of these complexes. The process enables detection of both the qualitative and quantitative state of protein complexes, including the identification of activated members with various covalent modifications such as phosphorylation, acetylation, glycosylation, and others.
The problem addressed is that current methods for exploring PPI, including mass spectrometry-based analyses and other biochemical assays, require large sample inputs and extensive sample purification, making them labor-intensive and unsuitable for high-throughput, especially when working with clinical samples that have limited cell numbers. The invention solves this by providing a high-throughput, high-sensitivity method that requires fewer cells and allows simultaneous analysis of hundreds of samples and interactions.
The proposed Multinodal Protein Interactome Network Array (MPINA) combines serial Co-IPs with RPPA, enabling the exploration of complex protein networks and signaling pathways. The technique supports comprehensive PPI and signaling profiling, including qualitative, quantitative, and kinetic data, from minimal sample input. The method is adaptable to various sample types, including tumor cells and clinical tissue specimens, and allows the identification of protein drug targets for guiding therapeutic decisions.
Claims Coverage
There is one independent claim, which establishes several inventive features for methods of treatment involving protein complex immunoprecipitation and analysis.
Method of treatment using protein complex immunoprecipitation and reverse phase protein array
A method involving: 1. Obtaining a tissue sample from a patient and generating a lysate from it. 2. Performing a protein complex immunoprecipitation by attaching a first antibody to the lysate to isolate a protein complex immunoprecipitate. 3. Performing a reverse phase protein array (RPPA) on this immunoprecipitate using a second and a third antibody, which are different from the first antibody used for immunoprecipitation. 4. Identifying a protein complex. 5. Determining whether the protein complex comprises a known protein drug target. 6. Treating the patient with a drug that interacts with the known protein drug target. The steps explicitly require that the antibody used for immunoprecipitation is different from those used in the RPPA.
The independent claim covers a method for identifying protein complexes and drug targets from a patient's tissue using sequential antibody-based immunoprecipitation and protein array technologies to guide patient treatment.
Stated Advantages
The method is high-throughput, allowing hundreds of samples and interactions to be analyzed simultaneously.
It has high sensitivity and can capture functional, multiplexed protein-protein interactions using very small numbers of cells.
It enables the capture of qualitative, quantitative, and kinetic data about protein-protein interactions.
The invention reduces the need for extensive sample purification compared to other techniques.
It can be used on clinical samples, including those with limited cellularity, such as tissue biopsies.
Documented Applications
Identifying protein-protein interactions and complexes in any cell type, including human samples.
Exploring protein networks and signaling pathway engagement in diseases such as cancer.
Guiding treatment selection for patients based on identification of protein drug targets in tissue samples.
Developing predictive or prognostic tests for clinical use in disease management.
Serving as a tool in the drug discovery process to identify targetable protein-protein interactions.
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