DETECTION METHOD FOR PROTEIN-PROTEIN INTERACTIONS USING STABLE ISOTOPE AMINO ACID LABELING IN CELL CULTURE

摘要

A method for detecting protein regions affected by protein-protein interactionsis provided, the method comprising: (a)providing a sample of a selected protein in a monomeric state, wherein the protein is expressed in cells cultured in a first form of a selected amino acid, and providing a sample of the selected protein in a state where it is bound to an interaction partner, wherein the bound protein is expressed in cells cultured in a second form of the selected amino acid, wherein the first form of amino acid and second form of amino acid differ in their molecular weight due to the use of stable isotopes in one or both of these forms of theselected amino acid; (b)treating the two samples obtained in step (a) with a modification reagent, which reagent modifies amino acidsthat are accessibleon the surface of the protein; (c)subjecting the reagent treated protein samples obtained in step (b) to proteolytic cleavage to generate reagent treated peptides; and (d)carrying out mass spectrometry on the reagent treated peptides obtained in step (c) to obtain mass spectra for the peptides.