Methods and compositions for detecting the activation state of multiple proteins in single cells

Abstract

The invention provides methods and compositions for simultaneously detecting the activation state of a plurality of proteins in single cells using flow cytometry. The invention further provides methods and compositions of screening for bioactive agents capable of coordinately modulating the activity of a plurality of proteins in single cells. The methods and compositions can be used to determine the protein activation profile of a cell for predicting or diagnosing a disease state, and for monitoring treatment of a disease state.

1 - 9 . (canceled) 
     
     
         10 . A method of detecting the activation state of at least a first activatable protein in single cells, said method comprising the steps of:
 a) providing a population of cells comprising at least said first activatable protein;   b) contacting said population of cells with a plurality of substrates; wherein said plurality of substrates comprise at least a first substrate that is capable of being modified by a corresponding isoform of said first activatable protein in said population of cells; and   c) using flow cytometry to detect the modification of said first substrate in single cells of said population of cells, wherein said modification is indicative of a specific activation state of said first activatable protein.   
     
     
         11 . The method according to  claim 10 , wherein said population of cells further comprises a second activatable protein, wherein said plurality of substrates further comprise a second substrate that is capable of being modified by a corresponding isoform of said second activable protein in said population of cells and, wherein step c) further comprises using said flow cytometry to detect the modification of said second substrate in single cells of said population of cells, wherein said modification of said second substrate is indicative of a specific activation state of said second activatable protein. 
     
     
         12 . A method of detecting a protein activation state profile of single cells based on the activation state of at least a first activatable protein in said cells, said method comprising the steps of:
 a) providing a population of cells comprising at least said first activatable protein;   b) contacting said population of cells with a plurality of substrates; wherein said plurality of substrates comprise at least a first substrate that is capable of being modified by a corresponding isoform of said first activatable protein in said population of cells;   c) contacting said population of cells with a plurality of activation state-specific antibodies, wherein said activation state-specific antibodies comprise at least one first activation state-specific antibody that is capable of binding to a corresponding isoform of said first activatable protein in said population of cells; and   d) using flow cytometry to simultaneously detect:   i) said binding of said first activation state-specific antibody in single cells of said population of cells, wherein said binding of said first activation state-specific antibody is indicative of a specific activation, state of said first activatable protein; and   ii) the modification of said first substrate said single cells, wherein said modification is indicative of said specific activation state of said first activatable protein.   
     
     
         13 - 32 . (canceled)