Affinity capture of peptides by microarray and related methods

Abstract

The invention provides methods of detecting polypeptides in a sample. The method can include the steps of cleaving polypeptides in a test sample to generate peptides; adding a predetermined amount of isotopically labeled peptide standards to the cleaved test sample, wherein the peptide standards correspond to peptides cleaved with the same reagent used to cleave the test sample; contacting the cleaved test sample containing peptide standards with an array of immobilized binding agents specific for the peptide standards; washing the array to remove unbound peptides, thereby retaining affinity captured sample peptides and standard peptides; analyzing the affinity captured peptides using mass spectrometry; and determining the presence of bound test peptides and standard peptides. The method can further include the step of quantifying the amount of the test peptides by comparing the ratio of test peptide to corresponding standard peptide.

1 - 15 . (canceled) 
     
     
         16 . A method of quantifying polypeptides in a sample, comprising:
 (a) cleaving said polypeptides to produce cleaved sample peptides;   (b) adding a predetermined amount of isotopically labeled standard peptides to said cleaved sample peptides, wherein said standard peptides correspond to said cleaved sample peptides;   (c) contacting said cleaved sample peptides containing said peptide standards with an array comprising immobilized binding agents, thereby generating immobilized sample peptides and standard peptides;   (d) removing unbound peptides, while retaining immobilized sample peptides and standard peptides; and   (e) using mass spectrometry to quantify immobilized sample peptides by determining the ratio of immobilized sample peptides to corresponding immobilized standard peptides, wherein the quantity of said immobilized sample peptides is indicative of the amount of uncleaved polypeptides in the sample.   
     
     
         17 . The method of  claim 16 , wherein said polypeptides are cleaved with a protease. 
     
     
         18 . The method of  claim 17 , wherein the protease is trypsin. 
     
     
         19 . The method of  claim 16 , wherein said binding agents are aptamers. 
     
     
         20 . The method of  claim 16 , wherein said binding agents are antibodies. 
     
     
         21 . The method of  claim 16 , wherein said isotopically labeled standard peptides are labeled with a stable isotope. 
     
     
         22 . The method of  claim 16 , wherein said sample peptides and standard peptides are differentially isotopically labeled with an isotope tag. 
     
     
         23 . The method of  claim 22 , wherein one of said isotope tags comprises deuterium. 
     
     
         24 . The method of  claim 16 , wherein said standard peptides are generated by chemical synthesis. 
     
     
         25 . The method of  claim 16 , wherein said standard peptides