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 - 32 . (canceled) 
     
     
         33 . A method comprising:
 (a) providing a cell comprising a first and a second activatable element, wherein the first and second activatable elements are different from one another and each have at least an activated form and a non-activated form;   (b) contacting the cell with:   a first activation state-specific antibody that binds to and is specific for the activated form or the non-activated form of the first activatable element; and   a second activation state-specific antibody that binds to and is specific for the activated form or the non-activated form of the second activatable element,   wherein at least one of the first and second activation state-specific antibodies is labeled, and wherein the first and second activation state-specific antibodies are distinguishably detectable from one another; and   (c) detecting the binding of the first and second activation state-specific antibodies to their corresponding activatable elements in the cell, wherein said detecting comprises one or more of: flow cytometry; fluorescence microscopy; fluorescence, luminescence, ultraviolet, and/or visible light detection using a plate reader; high performance liquid chromatography (HPLC); and mass spectrometry.   
     
     
         34 . The method according to  claim 33 , wherein said detecting comprises flow cytometry. 
     
     
         35 . The method according to  claim 33 , wherein said detecting comprises (i) fluorescence microscopy; (ii) fluorescent, ultraviolet, and/or visible light detection using a plate reader; (iii) high performance liquid chromatography (HPLC); or (iv) mass spectrometry. 
     
     
         36 . The method according to  claim 33 , wherein at least one of the activatable elements is an activatable protein. 
     
     
         37 . The method according to  claim 36 , wherein the first activatable element is a protein within the MAPK, AKT, STAT, NFkB, PKC or WNT signaling pathways and the second activatable element is a protein within the MAPK, AKT, STAT, NFkB, PKC or WNT signaling pathways. 
     
     
         38 . The method according to  claim 36 , wherein at least one of the activatable elements is a kinase or a caspase. 
     
     
         39 . The method according to  claim 33 , wherein the activated form of either or both of the first and second activatable elements is a phosphorylated form. 
     
     
         40 . The method according to  claim 33 , wherein the activated form of either or both of the first and second activatable elements is created through cleavage of the non-activated form. 
     
     
         41 . The method according to  claim 33 , wherein the activated form of either or both of the first and second activatable elements is created through a covalent addition, structural change, or conformational change. 
     
     
         42 . The method according to  claim 33 , wherein the activated form of either the first or second activatable element is a phosphorylated or a cleaved product of either the first or second activatable element. 
     
     
         43 . The method according to  claim 33 , wherein step (a) further comprises contacting the provided cell with an agent selected from: a cytokine, an interleukin, a hormone, a bioactive agent, a receptor ligand, and a therapeutic agent. 
     
     
         44 . The method according to  claim 33 , wherein at least one of the first and second activation state-specific antibodies is labeled with a directly detectable label. 
     
     
         45 . The method according to  claim 33 , wherein at least one of the activation state-specific antibodies is a peptide comprising a recognition structure. 
     
     
         46 . The method according to  claim 33 , wherein at least one of the activatable elements is a lipid. 
     
     
         47 . The method according to  claim 46 , wherein phosphatidylinositol 3, 4, 5 triphosphate (PIP3) and phosphatidylinositol 4, 5 bisphosphate (PIP2) are the activated and non-activated forms of the activatable element that is a lipid. 
     
     
         48 . A system for detecting the activation state of a cell, comprising:
 an apparatus comprising a detector configured to detect, in a cell, by mass spectrometry, the binding of a first activation state-specific antibody that binds to and is specific for an activated form or a non-activated form of a first activatable element in the cell, and the binding of a second activation state-specific antibody that binds to and is specific for an activated form or a non-activated form of a second activatable element in the cell, wherein: (i) the first and second activatable elements are different from one another; (ii) at least one of the first and second activation state-specific antibodies is labeled; and (iii) the first and second activation state-specific antibodies are distinguishably detectable from one another, and   an automated liquid handling system.   
     
     
         49 . The system according to  claim 48 , wherein the liquid handling system is completely automated. 
     
     
         50 . The system according to  claim 48 , wherein the liquid handling system is partially automated. 
     
     
         51 . The system according to  claim 48 , wherein the liquid handling system comprises one or more of: (i) a robotic arm, (ii) a plate handler, (iii) an automated lid or cap handler, (iv) a tip assembly for sample distribution, (v) a washable tip assembly for sample distribution, (vi) a multi-well loading block, (vii) a 96 well loading block, (viii) a cooled reagent rack, (ix) a stacking tower for plates and tips, and (x) a computer system. 
     
     
         52 . The system according to  claim 48 , wherein the liquid handling system provides for one or more manipulations selected from: aspiration, dispensing, mixing, diluting, washing, accurate volumetric transfers, retrieving and discarding of pipet tips, repetitive pipetting of identical volumes, replication of microplate samples, high-density transfers, and full-plate serial dilutions. 
     
     
         53 . The system according to  claim 48 , comprising a computer system comprising a module configured to determine the activation state of the first and second activatable elements in the cell based on the detection of binding of the differentially detectable first and second activation state-specific antibodies to their corresponding activatable elements in the cell. 
     
     
         54 . The system according to  claim 53 , comprising software configured to determine the activation state of the cell based on the determined activation states of the first and second activatable elements in the cell. 
     
     
         55 . The method according to  claim 48 , wherein both the first and second activation state-specific antibodies are labeled with distinguishably detectable labels. 
     
     
         56 . The system according to  claim 48 , wherein the apparatus comprises a sample chamber comprising a composition comprising a cell, wherein the cell comprises the first and second activation state-specific antibodies. 
     
     
         57 . The system according to  claim 48 , wherein the activated form of either or both of the first and second activatable elements is a phosphorylated form. 
     
     
         58 . The system according to  claim 48 , wherein the activated form of either or both of the first and second activatable elements is created through cleavage of the non-activated form. 
     
     
         59 . The system according to  claim 48 , wherein at least one of the activatable elements is an activatable protein 
     
     
         60 . The system according to  claim 48 , wherein at least one of the activation state-specific antibodies is a peptide comprising a recognition structure. 
     
     
         61 . The system according to  claim 48 , wherein at least one of the activatable elements is a lipid. 
     
     
         62 . The system according to  claim 61 , wherein phosphatidylinositol 3, 4, 5 triphosphate (PIP3) and phosphatidylinositol 4, 5 bisphosphate (PIP2) are the activated and non-activated forms of the activatable element that is a lipid.