Triazine derivatives as universal peptide isotope tag reagents (U-PIT)

Abstract

Compounds, compositions, methods for sequencing proteins and peptides, and methods for identifying proteins and peptides in a mixture, are disclosed. Compounds of formula A-B—C wherein A is a nucleophilic reactive group, B is a detectable moiety capable of being isotopically labeled, and C is a charge replacement group, are used to label the peptides at the N-terminus or the C-terminus. The tagged peptides can then be analyzed by mass spectroscopy.

1 . A compound of formula:  
       A-B—C  
     wherein: 
 A is a nucleophilic reactive group;  
 B is a detectable moiety capable of being isotopically labeled; and  
 C is a charge replacement group.  
 
   
   
       2 . The compound of  claim 1 , wherein A comprises:  
     
       
         
         
             
             
         
       
       wherein  
       G is selected from the group consisting of succinimide, maleimidie, glutarimide, isatin, phthalimide and halo-acetone;  
       X 1  and X 3  are independently selected from the group consisting of CR 1 R 2 , S, and NR 3  wherein R 1 , R 2 , R 3  are independently hydrogen or lower alkyl; and  
       X 2  is a direct bond or is selected from the group consisting of CR 1 R 2 , S, and NR 3  wherein R 1 , R 2 , R 3  are independently hydrogen or lower alkyl.  
     
   
   
       3 . The compound of  claim 2 , wherein G is succinimide.  
   
   
       4 . The compound of  claim 2 , wherein G is glutarimide.  
   
   
       5 . The compound of  claim 2 , wherein X 1  and X 3  are O.  
   
   
       6 . The compound of  claim 5 , wherein X 2  is O.  
   
   
       7 . The compound of  claim 5 , wherein X 2  is a direct bond.  
   
   
       8 . The compound of  claim 1 , wherein A is:  
     
       
         
         
             
             
         
       
     
   
   
       9 . The compound of  claim 1 , wherein A is:  
     
       
         
         
             
             
         
       
     
   
   
       10 . The compound of  claim 1 , wherein A is:  
     
       
         
         
             
             
         
       
     
   
   
       11 . The compound of  claim 1 , wherein A is:  
     
       
         
         
             
             
         
       
     
   
   
       12 . The compound of  claim 1 , wherein A is:  
     
       
         
         
             
             
         
       
       wherein X is a halogen selected from the group consisting of Cl, Br, and I.  
     
   
   
       13 . The compound of  claim 1 , wherein B is selected from the group consisting of lowerl alkyl, ether, aryl, and heteroaryl.  
   
   
       14 . The compound of  claim 13 , wherein B is ether.  
   
   
       15 . The compound of  claim 14 , wherein ether is polyethyleneglycol.  
   
   
       16 . The compound of  claim 13 , wherein B is heteroaryl.  
   
   
       17 . The compound of  claim 16 , wherein the heteroaryl is selected from the group consisting of substituted or unsubstitued 1,3,5-triazine, 1,2,5-triazine, 1,2,3-triazine, pyridine, imidazole, and thiophene.  
   
   
       18 . The compound of  claim 1 , wherein B comprises the formula:  
     
       
         
         
             
             
         
       
       wherein X′ and R′ taken together are H or halogen;  
       X′ and X″ are independently NH, O, or S; and  
       R, R′ and R″ are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, and (CH 2 CH 2 O) n  where n is an integer between 1 and 5.  
     
   
   
       19 . The compound of  claim 18 , wherein X and X′ are NH, R is CH 2 CH 2 , and R′ is ethyl.  
   
   
       20 . The compound of  claim 18 , wherein X and X′ are NH, R is CH 2 CH 2 OCH 2 CH 2 , and R′ is ethyl.  
   
   
       21 . The compound of  claim 18 , wherein at least one atom in B is replaced with its isotope.  
   
   
       22 . The compound of  claim 21 , wherein the isotope is selected from the group consisting of  2 H,  3 H,  13 C,  14 C,  32 P,  34 S,  35 S,  36 Cl,  37 Cl,  18 O,  15 N,  81 Br,  123 I,  125 I and  131 I.  
   
   
       23 . The compound of  claim 18 , wherein all atoms in B are replaced with their isotopes.  
   
   
       24 . The compound of  claim 18 , wherein mass difference between the isotopically labeled and unlabeled compound is at least 2 mass units.  
   
   
       25 . The compound of  claim 24 , wherein the mass difference is between 3 and 15 mass units.  
   
   
       26 . The compound of  claim 1 , wherein C is a quaternary nitrogen derivative.  
   
   
       27 . The compound of  claim 26 , wherein the quaternary nitrogen comprises the formula:  
     
       
         
         
             
             
         
       
       wherein R 4 , R 5 , and R 6  are independently selected from the group consisting of hydrogen, lower alkyl and aryl.  
     
   
   
       28 . The compound of  claim 27 , wherein R 5 , and R 6  are methyl.  
   
   
       29 . The compound of  claim 28 , wherein R 4  is methyl.  
   
   
       30 . The compound of  claim 28 , wherein R 4  is tert-butyl.  
   
   
       31 .- 49 . (canceled)  
   
   
       50 . A kit for detecting the presence of a plurality of target peptides in a sample, the kit comprising a compound of formula A-B—C wherein A is a nucleophilic reactive group, B is a detectable moiety capable of being isotopically labeled, and C is a charge replacement group; and written instructions.  
   
   
       51 .- 55 . (canceled)