Quantitative peptide or protein assay

Abstract

Peptide and/or protein quantitation methods, kits, and compositions, particularly useful for mass spectrometry, are provided herein based on a bathocuproine-based composition complex such as bathocuproinedisulfonic acid disodium salt hydrate complex. The methods are one-step rapid absorbance methods using small sample volumes. They produce a robust signal with high signal to background ratio and accurately quantitate even complex peptide mixtures with low variability and high sensitivity.

What is claimed is: 
     
         1 - 23 . (canceled) 
     
     
         24 . A kit for protein or peptide quantitation comprising:
 (a) a complex having the formula:   
       
         
           
           
               
               
           
         
         wherein
 each of R 1  and R 2  is independently chosen from C 1 -C 6  straight and branched alkyls, C 6 -C 20  aryl, alkylaryl, and arylalkyls; 
 each of R 3  and R 4  is independently chosen from the group consisting of hydrogen; sulfonate salts chosen from sodium, potassium, and lithium; phosphonate salts chosen from sodium, potassium, and lithium; and carboxylate salts chosen from sodium, potassium, and lithium; and 
 each of R 5  and R 6  is 
 
       
       
         
           
           
               
               
           
         
       
       wherein R 7  is independently chosen from the group consisting of hydrogen; sulfonate salts chosen from sodium, potassium, and lithium; phosphonate salts chosen from sodium, potassium, and lithium; and carboxylate salts chosen from sodium, potassium, and lithium; 
       with the proviso that at least one of R 3 , R 4 , and R 7  is not H;
 (b) acetonitrile; and 
 (c) copper sulfate. 
 
     
     
         25 . The kit of  claim 24 , wherein the complex is present at a concentration ranging from about 0.04 M to about 0.1 M. 
     
     
         26 . The kit of  claim 24 , wherein each of R1 and R2 is independently chosen from methyl, ethyl, propyl, butyl, and phenyl. 
     
     
         27 . The kit of  claim 24 , wherein the complex has the formula: 
       
         
           
           
               
               
           
         
       
     
     
         28 . The kit of  claim 24 , wherein the acetonitrile is present at a concentration ranging from about 10% to about 50%. 
     
     
         29 . The kit of  claim 28 , wherein the acetonitrile is present at a concentration ranging from about 10% to about 30%. 
     
     
         30 . The kit of  claim 24 , wherein the copper sulfate is present at a concentration ranging from about 0.25 mM to about 0.5 mM. 
     
     
         31 . The kit of  claim 24 , further comprising tartrate. 
     
     
         32 . The kit of  claim 31 , wherein the tartrate is present at a concentration ranging from about 5.7 mM to about 22.7 mM. 
     
     
         33 . The kit of  claim 31 , where the tartrate is sodium tartarate. 
     
     
         34 . The kit of  claim 24 , further comprising sodium bicarbonate. 
     
     
         35 . A composition for protein or peptide quantitation comprising:
 (a) a complex having the following general formula:   
       
         
           
           
               
               
           
         
         wherein
 each of R 1  and R 2  is independently chosen from C 1 -C 6  straight and branched alkyls, C 6 -C 20  aryl, alkylaryl, and arylalkyls; 
 each of R 3  and R 4  is independently chosen from the group consisting of hydrogen; 
 
         sulfonate salts chosen from sodium, potassium, and lithium; phosphonate salts chosen from sodium, potassium, and lithium; and carboxylate salts chosen from sodium, potassium, and lithium; and
 each of R 5  and R 6  is 
 
       
       
         
           
           
               
               
           
         
       
       where R 7  is independently chosen from the group consisting of hydrogen; sulfonate salts chosen from sodium, potassium, and lithium; phosphonate salts chosen from sodium, potassium, and lithium; and carboxylate salts chosen from sodium, potassium, and lithium; 
       with the proviso that at least one of R 3 , R 4 , and R 7  is not H;
 (b) acetonitrile; and 
 (c) copper sulfate. 
 
     
     
         36 . The composition of  claim 35 , wherein the complex is present at a concentration ranging from about 0.04 M to about 0.1 M. 
     
     
         37 . The kit of  claim 35 , wherein each of R 1  and R 2  is independently chosen from methyl, ethyl, propyl, butyl, and phenyl. 
     
     
         38 . The composition of  claim 35 , wherein the complex has the formula: 
       
         
           
           
               
               
           
         
       
     
     
         39 . The composition of  claim 35 , wherein the acetonitrile is present at a concentration ranging from about 10% to about 50%. 
     
     
         40 . The composition of  claim 39 , wherein the acetonitrile is present at a concentration ranging from about 10% to about 30%. 
     
     
         41 . The composition of  claim 35 , wherein the copper sulfate is present at a concentration ranging from about 0.25 mM to about 0.5 mM. 
     
     
         42 . The composition of  claim 35 , further comprising tartrate. 
     
     
         43 . The composition of  claim 42 , wherein the tartrate is present at a concentration ranging from about 5.7 mM to about 22.7 mM. 
     
     
         44 . The composition of  claim 42 , where the tartrate is sodium tartrate. 
     
     
         45 . The composition of  claim 34 , further comprising sodium bicarbonate. 
     
     
         46 . The composition of  claim 35 , wherein the pH ranges from about 12 to about 13. 
     
     
         47 . A kit for protein or peptide quantitation comprising:
 (a) a complex having the formula:   
       
         
           
           
               
               
           
         
         (b) acetonitrile; and 
         (c) copper sulfate.