Protein splice variant / isoform discrimination and quantitative measurements thereof

Abstract

The invention relates to methods, reagents and apparatus for detecting protein isoforms (e.g., those due to alternative splicing, or different disease protein isoforms or degradation products) in a sample, including using combinations of capture agents, each combination being unique to the splicing variant to be detected/measured.

1 . A method for detecting a protein isoform in a sample, the method comprising:
 (1) fragmenting the protein isoform in the sample to produce a peptide fragment comprising a first epitope and a second epitope.   (2) contacting said peptide fragment with a pair of first and second binding agents which bind respectively to said first and second epitopes, the binding of the combination of the binding agents being unambiguously indicative of the presence of the isoform in the sample, and,   (3) detecting the binding of said first and second binding agents to said peptide fragment as an indication of the presence of said isoform in the sample.   
     
     
         2 . The method of  claim 1 , wherein step (1) is done by digestion using a protease. 
     
     
         3 . The method of  2 , wherein said protease is trypsin or LysC. 
     
     
         4 . The method of  claim 1 , wherein said protein isoform is an alternative splicing isoform. 
     
     
         5 . The method of  claim 4 , wherein the alternative splicing isoform is one of at least two alternative splicing isoforms present in the sample. 
     
     
         6 . The method of  claim 4 , wherein the alternative splicing isoform is the only isoform present in the sample. 
     
     
         7 . The method of  claim 1 , wherein the sample is pre-treated by purification and/or denaturation prior to step (2). 
     
     
         8 . The method of  claim 1 , wherein the first and/or the second binding agents are antibodies or a functional antibody fragment thereof. 
     
     
         9 . The method of  claim 1 , further comprising quantitating the binding of said first or second binding agents to said peptide fragment to determine the amount and/or concentration of said isoform in the sample. 
     
     
         10 . The method of  claim 1 , wherein at least one of said binding agents is detectably labeled. 
     
     
         11 . The method of  claim 10 , wherein the binding agents is detectably labeled by a fluorescent label. 
     
     
         12 . The method of  claim 1 , wherein at least one of said binding agents is immobilized on a solid support. 
     
     
         13 . The method of  claim 12 , wherein the solid support is an array. 
     
     
         14 . The method of  claim 1 , wherein at least one of said binding agents is detectably labeled and another is immobilized on a solid support. 
     
     
         15 . The method of  claim 1 , wherein neither said first epitope nor said second epitope span a junction between the expression products of different exons. 
     
     
         16 . The method of  claim 1 , wherein both said first epitope and said second epitope reside in the expression product of one exon. 
     
     
         17 . The method of  claim 16 , wherein said expression product of said one exon is unique to the isoform. 
     
     
         18 . The method of  claim 1 , wherein the binding of one of said binding agents to one of said first and second epitopes is not unambiguously indicative of the presence of said protein isoform in the sample. 
     
     
         19 . The method of  claim 1 , for multiplexed detection of plural different isoforms of said protein in said sample comprising a mixture of proteins, the method comprising:
 (1) fragmenting proteins in the sample to produce plural peptide fragments, which comprise first and second epitope pairs and at least portions of the expression product of exon(s) encoding at least a portion of the respective proteins, the presence of said fragments being unambiguously indicative of the presence of the isoforms in the sample:   (2) contacting said peptide fragments with pairs of first and second binding agents which bind respectively to said first and second epitope pairs, and,   (3) detecting the binding as an indication of the presence of respective said isoforms in the sample.   
     
     
         20 . The method of  claim 19 , further comprising quantitating the binding of said binding agents to determine at least the relative quantity of at least two different isoforms in the sample. 
     
     
         21 . The method of  claim 19 , wherein detection of binding is effected by detecting optical signals generated by optical labels on one said binding agents to a said peptide captured at a selected position on a solid support.