Discovery and validation of cancer biomarkers using a protein analysis methodology to analyze specimens

Abstract

Methods are provided for the analysis, including the serial analysis, of very small samples of tissue. The methods utilize a nanofluidic proteomic immunoassay (NIA) to quantify total and low-abundance protein isoforms in a small amount of lysate. NIA detection accurately measure oncoprotein expression and activation in limited clinical specimens, including isoforms that differ in post-translational modifications, such as phosphorylation, and the like. The NIA detection method combines isoelectric protein focusing and antibody detection in a nanofluidic system.

1 . A method of screening for the discovery of disease associated biomarkers for diagnostic or therapeutic intervention, the method comprising;
 performing NIA on a cellular sample;   determining the presence of a biomarker of interest in said cellular sample;   comparing said biomarker in said cellular sample with a control tissue;   wherein biomarkers in said cellular sample represent molecular targets for therapeutic or diagnostic intervention.   
     
     
         2 . The method according to  claim 1 , wherein said sample is a biopsy sample. 
     
     
         3 . The method of  claim 2 , wherein the biopsy sample is a tumor cell sample of less than 100,000 cells. 
     
     
         4 . The method of  claim 3 , wherein the biomarker is an oncoprotein isoform. 
     
     
         5 . The method of  claim 4 , wherein the isoform differs from alternative isoforms of the same oncoprotein by phosphorylation. 
     
     
         6 . The method of  claim 5 , wherein the biomarker is ERK2, and the single phosphorylated form is down regulated in a CML sample from a patient that is response to a tyrosine kinase inhibitor. 
     
     
         7 . The method of  claim 6 , wherein the tyrosine kinase inhibitor is imatinib. 
     
     
         8 . The method according to  claim 4 , wherein the control tissue is a sample from the same tumor at a different time point. 
     
     
         9 . The method of  claim 8 , wherein multiple time points from a single tumor are compared. 
     
     
         10 . The method of  claim 1 , wherein the cellular sample was previously frozen. 
     
     
         11 . The method of  claim 1 , wherein said cellular sample comprises blood cells. 
     
     
         12 . The method of  claim 1 , wherein said cellular sample is maintained on ice for greater than 30 minutes prior to cell lysis. 
     
     
         13 . The method of  claim 1 , wherein said cellular sample is suspected of comprising kidney cancer cells, and said biomarker is % phospho-ERK. 
     
     
         14 . The method of  claim 1 , wherein said cellular sample is suspected of comprising head and neck cancer cells and said biomarker is % ppERK1.