Chemical reagents and methods for detection and quantification of proteins in complex mixtures

Abstract

The invention provides a reagent comprising an affinity tag, a detectable moiety, a linker, an isotope tag and a reactive group. The invention also provides methods of using a reagent of the invention. The methods can be used to label a polypeptide in a sample by contacting a sample with a reagent of the invention under conditions allowing the reactive group to bind to one or more polypeptides in the sample. The invention additionally provides methods of isolating, identifying and quantifying a polypeptide in a sample. The invention further provides methods of diagnosing a disease using a reagent of the invention.

1 . A reagent comprising an affinity tag, a detectable moiety, a linker, an isotope tag and a reactive group. 
     
     
         2 . The reagent of  claim 1 , wherein the detectable moiety is a fluorophore or chromophore. 
     
     
         3 . The reagent of  claim 1 , wherein the detectable moiety is radioactive. 
     
     
         4 . The reagent of  claim 3 , wherein the detectable moiety comprises a radioactive atom selected from the group consisting of 3H, 14C, 32P, 35S and 125I. 
     
     
         5 . The reagent of  claim 1 , wherein said linker is cleavable. 
     
     
         6 . The reagent of  claim 5 , wherein said linker comprises a photocleavable moiety, a chemically cleavable moiety, or an enzymatically cleavable moiety. 
     
     
         7 . The reagent of  claim 1 , wherein said affinity tag is selected from the group consisting of biotin or a derivative thereof, glutathione, maltose, poly(His), and an epitope tag. 
     
     
         8 . The reagent of  claim 1 , wherein said reactive group is selected from the group consisting of a sulfhydryl reactive group, an amine reactive group, and a carboxyl reactive group. 
     
     
         9 . The reagent of  claim 1 , said reagent having the general formula: 
       
         
           
           
               
               
           
         
       
       wherein A is an affinity tag, D is a detectable moiety, L is a linker, T is an isotope tag, and R is a reactive group. 
     
     
         10 - 16 . (canceled) 
     
     
         17 . A kit comprising the reagent of  claim 1 . 
     
     
         18 - 24 . (canceled) 
     
     
         25 . A method of labeling a polypeptide in a sample, comprising contacting a sample with the reagent of  claim 1  under conditions allowing the reactive group to bind to one or more polypeptides in said sample. 
     
     
         26 - 32 . (canceled) 
     
     
         33 . A method of isolating a polypeptide in a sample, comprising the steps of:
 (a) contacting a sample with the reagent of  claim 1  under conditions allowing the reactive group to bind to and react with one or more polypeptides in said sample, thereby tagging one or more polypeptides with said reagent;   (b) resolving the polypeptides in said sample;   (c) visualizing the polypeptides tagged with said reagent;   (d) contacting said tagged polypeptides with a capture moiety for said affinity tag; and   (e) isolating said tagged polypeptides.   
     
     
         34 - 41 . (canceled) 
     
     
         42 . A method of identifying a polypeptide in a sample, comprising the steps of:
 (a) contacting a sample with the reagent of  claim 1  under conditions allowing the reactive group to bind to and react with one or more polypeptides in said sample, thereby tagging one or more polypeptides with said reagent;   (b) resolving the polypeptides in said sample;   (c) visualizing the one or more polypeptides tagged with said reagent;   (d) contacting said one or more tagged polypeptides with a capture moiety for said affinity tag;   (e) isolating said one or more tagged polypeptides;   (f) cleaving the linker of said reagent to release said one or more tagged polypeptides; and   (g) identifying a released tagged polypeptide.   
     
     
         43 . The method of  claim 42 , wherein said identifying step is performed using mass spectrometry. 
     
     
         44 . The method of  claim 43 , further comprising quantifying the amount of said identified polypeptide. 
     
     
         45 - 52 . (canceled) 
     
     
         53 . A method of identifying a polypeptide in a sample, comprising the steps of:
 (a) contacting a sample with the reagent of  claim 1  under conditions allowing the reactive group to bind to and react with one or more polypeptides in said sample, thereby labeling one or more polypeptides with said reagent;   (b) cleaving the polypeptides in said sample to generate peptide fragments;   (c) adding one or more peptide standards to said sample, wherein said peptide standards correspond to peptides generated from cleaving sample polypeptides as in step (b) and are labeled with an isotopically distinct version of said isotope tag;   (d) resolving the labeled peptides in said sample;   (e) visualizing the labeled peptides;   (f) contacting said labeled peptides with a capture moiety for said affinity tag;   (g) isolating said labeled peptides;   (h) cleaving the linker of said reagent to release said isolated peptides; and   (i) identifying a released peptide.   
     
     
         54 - 64 . (canceled) 
     
     
         65 . A method of diagnosing a disease, comprising the steps of:
 (a) contacting a sample from a patient with the reagent of  claim 1  under conditions allowing the reactive group to bind to and react with one or more polypeptides in said sample, thereby labeling one or more polypeptides with said reagent;   (b) cleaving the polypeptides in said sample to generate peptide fragments;   (c) adding one or more peptide standards to said sample, wherein said peptide standards correspond to peptides generated from cleaving sample polypeptides as in step (b), are labeled with an isotopically distinct version of said isotope tag, and are derived from a polypeptide exhibiting aberrant expression in said disease;   (d) resolving the labeled peptides in said sample;   (e) visualizing the labeled peptides;   (f) contacting the labeled peptides with a capture moiety for said affinity tag;   (g) isolating said labeled peptides;   (h) cleaving the linker of said reagent to release said isolated peptides;   (j) quantifying said released peptides; and   (k) comparing the amount of sample peptide to standard peptide in said released peptides, wherein the amount of sample peptide is correlated with the diagnosis of said disease.   
     
     
         66 . The method of  claim 65 , wherein said disease is muscular dystrophy. 
     
     
         67 . The method of  claim 66 , wherein said disease is Duchenne muscular dystrophy or Becker muscular dystrophy. 
     
     
         68 . The method of  claim 65 , wherein said disease is prostate cancer. 
     
     
         69 - 78 . (canceled)