Quantification of Protein-Protein Interaction of Membrane Proteins Using High-Mass Mass Spectrometry

Simple SummaryContent extracted from patent full text and abstract with AI.

This invention presents a high-throughput, mass spectrometry-based method for quantifying the interactions between membrane proteins (such as G-protein-coupled receptors, GPCRs) and their partner proteins. The process stabilizes protein complexes by introducing a crosslinker and then uses high-mass MALDI mass spectrometry to detect and quantify both native and transient protein complexes. The method enables detailed analysis of how ligands or compounds affect the ability of membrane proteins to interact with their partners, offering quantitative data on binding affinities and selectivity without the need for protein labeling or large sample amounts.

Use CasesContent extracted from patent full text and abstract with AI.

• Drug discovery: Screening and profiling the interaction strengths of potential drug compounds with GPCRs and other membrane proteins.
• Pharmaceutical research: Studying ligand-induced conformational changes and allosteric effects on membrane proteins for the development of more selective drugs.
• Biochemistry and molecular biology: Investigating the molecular mechanisms of protein-protein interactions in signaling pathways involving membrane proteins.
• GPCR functional studies: Elucidating biased signaling and coupling selectivity between GPCRs and various G proteins, arrestins, or other partners.
• Academic research: Supporting studies on membrane receptor structure, function, and complex formation dynamics.

BenefitsContent extracted from patent full text and abstract with AI.

• Enables direct, quantitative measurement of protein-protein interactions involving membrane proteins, which are traditionally difficult to study.
• High sensitivity and throughput—requires very small sample amounts and allows rapid analysis of hundreds of samples per plate.
• Does not require labeling, immobilization, or extensive modification of proteins, preserving their natural activity and complex integrity.
• Provides detailed affinity and selectivity profiles, supporting deeper understanding of allosteric modulation and signaling mechanisms.
• Broad tolerance to buffers, salts, detergents, or lipids, making the method versatile for various biochemical and pharmaceutical assays.
• Facilitates high-throughput drug screening and profiling, supporting more efficient and accurate drug development processes.

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Inventors & Applicants

Patent Abstract

G-protein-coupled receptors (GPCRs) are important pharmaceutical targets for the treatment of a broad spectrum of diseases. Although there are structures of GPCRs in their active conformation with bound ligands and G-proteins, the detailed molecular interplay between the receptors and their signaling partners remains challenging to decipher. To address this, a high-throughput quantification method to quantify protein-protein interaction of membrane proteins is disclosed, comprising the steps of: a) providing a solution comprising a membrane protein, such as a GPCR, in the absence or presence of a chemical compound / compounds or ligands that can bind to or modulate the membrane protein;; b) adding a partner protein to the solution and after a predetermined time interval adding additionally a crosslinker that reacts with proteins' surface amino acid, such as lysines or other amino acids depending on the specific crosslinker that is used, in order to form chemical linkages that stabilize compound complexes of biomolecules; c) detecting and quantifying stabilized native and transient complexes of the biomolecules and the non-interacting biomolecule counterparts by mass spectrometry using a reference peak, i.e. in terms of a normalization strategy, to investigate the binding ability of the partner protein to the membrane protein. Thus, this high-sensitivity, high-throughput mass spectrometry method interrogates the first stage of signal transduction. The membrane protein and partner protein complex formation is detected as a proxy for the effect of ligands on membrane protein conformation and on coupling selectivity. The method requires only very little probe amounts,