Method and apparatus for signal transduction pathway profiling

Abstract

An assay device for determining the presence of analytes in a cell lysate comprises a porous support member and a plurality of binding reagents arranged and immobilized at multiple reaction sites on the support member. The binding reagents are selected and arranged to assess the status of a selected cellular signal transduction pathway/protein-protein interactive network. In a further aspect, a method for assessing the status of a signal transduction pathway comprises generating a lysate of cells, the lysate retaining one or more pathway molecules present in one or more states and the pathway molecules reflecting signal transduction events taking place in the cells. The method further includes applying the lysate to an immobilized series of binding reagents which can discriminate the pathway molecules and their states. Binding events between the pathway molecules and the binding reagents are identified and the state of the selected signal pathway is determined.

1 . An assay device for use in determining the presence of a plurality of analytes in a lysate of a sample of cells, the device comprising:
 (a) a porous support member; and   (b) a plurality of binding reagents arranged and immobilized at a plurality of reaction sites on the support member, the binding reagents selected and arranged to assess the status of a selected protein-protein interaction network when the lysate is applied thereto.   
     
     
         2 . The device of  claim 1 , wherein the protein-protein interaction network is a signal transduction pathway. 
     
     
         3 . The device of  claim 2 , wherein the binding reagents are arranged to quantitatively assess the status of the selected signal transduction pathway. 
     
     
         4 . The device of  claim 2 , wherein each binding reagent selectively binds with one of a series of biological molecules, the signal transduction pathway comprising said series of biological molecules, the biological molecules selected from one or more proteins, a phosphorylated state of said one or more proteins, an activated state of said one or more proteins, and a binding partner of said one or more proteins. 
     
     
         5 . The device of  claim 1 , wherein the protein-protein interaction network is relevant to a disease state. 
     
     
         6 . The device of  claim 5 , wherein the disease state is selected from cancer, brain disease, cardiac disease, and an allergy. 
     
     
         7 . The device of  claim 2 , wherein the binding reagents are ordered to at least partially recapitulate a temporal or spatial status of molecular complex formations or networks of protein interaction which characterize the selected protein-protein interactive network. 
     
     
         8 . The device of  claim 7 , wherein the binding reagents are bound in an immobilized flow-through matrix such that a plurality of protein complexes comprising different combinations of the same molecules can be sorted to determine the relative proportion of at least one selected molecule within the protein complexes. 
     
     
         9 . The device of  claim 7 , wherein the protein-protein interactive network is branched. 
     
     
         10 . The device of  claim 7 , wherein the protein-protein interactive network is a signal transduction pathway. 
     
     
         11 . The device of  claim 10 , wherein the binding reagents are ordered to at least partially recapitulate the selected cellular signal transduction pathway in reverse temporal order. 
     
     
         12 . The device of  claim 10 , wherein the binding reagents are ordered to at least partially recapitulate the selected cellular signal transduction pathway in forward temporal order. 
     
     
         13 . The device of  claim 10 , wherein the binding reagents are ordered to at least partially recapitulate one of a signal pathway branch point and a connection to another pathway. 
     
     
         14 . The device of  claim 1 , which comprises a kit for providing information about the therapeutic, toxic, or diagnostic state of a cellular sample. 
     
     
         15 . A method for assessing the status of a selected signal transduction pathway in cells comprising:
 (a) generating a lysate of cells containing components of signal pathway proteins, the lysate retaining, at least in part, one or more pathway molecules, the pathway molecules present in one or more states selected from inactive, activated, activity altered, phosphorylated, cleaved, modified, and bound, the one or more pathway molecules reflecting signal transduction events taking place in the cells;   (b) applying the lysate to an immobilized series of binding reagents which discriminate the one or more pathway molecules and the one or more states thereof;   (c) identifying binding events between the pathway molecules and the binding reagents; and   (d) determining the state of the selected signal pathway.   
     
     
         16 . The method of  claim 15 , wherein the binding reagents are bound in an immobilized flow-through matrix such that protein complexes comprising different combinations of the same molecules can be sorted to determine the relative proportion of at least one chosen molecule within different complexes. 
     
     
         17 . The method of  claim 15 , wherein the immobilized binding reagents are arranged in a preselected arrangement and wherein the determining step comprises identifying a pattern of binding events based on said arrangement to form a pattern of binding events. 
     
     
         18 . A method for identifying proteins involved in cellular signaling or networks comprising:
 (a) exposing cells to one of a phosphatase inhibitor followed by a drug such that the phosphorylation state of one or more molecules is changed;   (b) analyzing patterns of groups of at least 2 phosphorylated molecules before and after exposure to the drug;   (c) exposing cells to a molecule which perturbs one or more pathways; and   (d) comparing the patterns before and after exposure of the cells to said molecule.   
     
     
         19 . The method of  claim 18 , further including the step of: (e) identifying therapeutic targets by evaluating whether a candidate compound either binds to a phosphorylated protein or perturbs the pattern of groups of at least 2 phosphorylated proteins. 
     
     
         20 . The method of  claim 18 , further comprising identifying one or both of drug-induced toxicity and therapeutic efficacy by evaluating one or more patterns of protein-protein interactions.