Patent ID: 8663928

Claim:
A method for detection in a homogeneous medium of a post-translational modification of a protein or peptide substrate catalyzed by a cell enzyme, wherein the post-translational modification is a reaction that takes place in intact living cells, wherein the cells comprise a heterologous expression vector coding for a fusion protein comprising the protein or peptide substrate and a first coupling domain, wherein the fusion protein also comprises a second coupling domain, different from the first coupling domain, said second coupling domain not being affected by the post-translational modification, said method comprising the following steps: (i) incubating the cells in the presence or in the absence of a compound to be tested capable of modulating the activity of said enzyme; (ii) distributing the cells incubated during step (i) in two different measurement media first and second measurement media; (iii) adding to a first measurement medium a first fluorescent compound that is a member of a first pair of FRET (Fluorescence Resonance Energy Transfer) partners, said first fluorescent compound being covalently bonded to a coupling agent capable of binding specifically to said first coupling domain present on the protein or peptide substrate; and said first pair of FRET partners emitting a FRET signal when the FRET partners are close to one another; (iv) adding to said first measurement medium a second fluorescent compound that is a member of this first pair of FRET partners, said second fluorescent compound being covalently bonded to a binding domain specific to the site of the protein or peptide substrate having undergone the post-translational modification and not binding to the non-modified protein or peptide substrate; (v) measuring the FRET signal emitted by the first measurement medium, this signal representing the quantity of protein or peptide substrate having undergone said post-translational modification; (vi) adding to a second measurement medium said first fluorescent compound that is a member of said first pair of FRET partners, said first fluorescent compound being covalently bonded to a coupling agent capable of binding specifically to said first coupling domain present on the protein or peptide substrate; (vii) adding to said second measurement medium a third fluorescent compound constituting with the first fluorescent compound a second pair of FRET partners, this third fluorescent compound being covalently bonded to a coupling agent capable of binding specifically to said second coupling domain, and said second pair of FRET partners emitting a FRET signal when the FRET partners are close to one another; (viii) measuring the FRET signal emitted by the second measurement medium, this signal representing the total quantity of protein or peptide substrate; and (ix) standardizing the signal measured in step (v) by the signal measured in step (viii), thereby detecting the post-translational modification of the protein or peptide substrate.