Methods and kits useful for the simplification of complex peptide mixtures

Abstract

Methods and reagents for obtaining simplified mixtures of peptides from a sample containing a number of peptides are disclosed. The methods entail the use of tagging moieties that include an amino-acid-specific reactive group (R). The tagging moieties “tag” peptides or proteins at specific amino acids (e.g., by reacting with an amino acid to form a covalent bond), ultimately allowing the isolation of peptides that contain those specific amino acids. Other methods entail the used of a reactive moiety (R P ) that comprises a reagent that selectively interacts with selected proteins, either covalently or noncovalently. For example, R P can be a natural ligand for a receptor that is to be tagged or a protein that interacts with a second protein that is to be tagged. It can be an enzymatic substrate or other element of molecular recognition such as an antibody, ATP, GTP, NAD, NADP, NADH, NADPH, ubiquitin, or structural analogs thereof.

1 . A reagent kit comprising: 
 a tagging moiety having the formula:      R-L-B    wherein R is a reactive group that reacts with peptides having an amino terminal serine or an amino terminal threonine, L is a linker group, and B is a group that can selectively bind a capture reagent    
   
   
       2 . The reagent kit of  claim 1  further comprising a proteolytic enzyme.  
   
   
       3 . The reagent kit of  claim 1  further comprising a capture reagent.  
   
   
       4 . The reagent kit of  claim 1  wherein R comprises —CO—NH—NH 2 .  
   
   
       5 . The reagent kit of  claim 1  wherein the tagging moiety is biotin hydrazide.  
   
   
       6 . The reagent kit of  claim 5  further comprising a capture reagent comprising avidin or streptavidin bound to a solid support; and D-biotin.  
   
   
       7 . The reagent kit of  claim 6  further comprising an oxidizing agent.  
   
   
       8 . The reagent kit of  claim 7  wherein the oxidizing agent is sodium metaperiodate and the kit further comprises an agent capable of quenching the oxidizing agent.  
   
   
       9 . The reagent kit of  claim 1  wherein L or R is isotopically labeled.  
   
   
       10 . A reagent kit comprising: 
 a tagging moiety having the formula:      R-L-M    wherein R is a reactive group that reacts with peptides comprising a selected amino acid, L is a linker group, and M is a magnetic particle that can be attracted by a magnetic force.    
   
   
       11 . The reagent kit of  claim 10  wherein R comprises: 
 (a) an amine reactive moiety,    (b) —CO—NH—NH 2 ,    (c) a thiol reactive moiety, or    
   
   
       12 . The reagent kit of  claim 10  wherein L is isotopically labeled.  
   
   
       13 . The reagent kit of  claim 10  wherein R is isotopically labeled.  
   
   
       14 . A reagent kit containing a moiety comprising: 
 a tagging moiety having the formula R-L-Sol-M R-L-Sol-L-M or R-Sol-L-M, wherein R is a reactive group that reacts with a peptides comprising a selected amino acid, L is a linker group, Sol is a hydrophilic polymer, and M is a magnetic particle that can be attracted by a magnetic force.    
   
   
       15 . The reagent kit of  claim 14  where R comprises: 
 (a) an amine reactive moiety,    (b) a thiol reactive moiety,    (c) —CO—NH—NH 2 ; or    (d) an enzymatic substrate or other noncovalent element of molecular recognition such as ATP, GTP, NAD, NADP, NADH, NADPH, ubiquitin, or structural analogs thereof.    
   
   
       16 . The kit of  claim 14  wherein R or L is isotopically labeled  
   
   
       17 . A reagent kit containing a moiety comprising: 
 a tagging moiety having the formula R-L-Sol-B or R-Sol-L-B,    wherein R is a reactive group that reacts with a peptides comprising a selected amino acid, L is a linker group, Sol is a hydrophilic polymer, and B is a group that can selectively bind to a capture reagent.    
   
   
       18 . The reagent of  claim 17  where R comprises: 
 (a) an amine reactive moiety,    (b) a thiol reactive moiety,    (c) —CO—NH—NH 2 ; or    (d) an enzymatic substrate or other noncovalent element of molecular recognition such as ATP, GTP, NAD, NADP, NADH, NADPH, ubiquitin, or structural analogs thereof.    
   
   
       19 . The reagent kit of  claim 17  wherein R or L is isotopically labeled  
   
   
       20 . A reagent kit containing a moiety comprising: 
 a tagging moiety having the formula R-L-Sol,    wherein R is a reactive group that reacts with a peptides comprising a selected amino acid, L is a linker group, and Sol is a hydrophilic polymer.    
   
   
       21 . The reagent kit of  claim 20  where R comprises: 
 (a) an amine reactive moiety,    (b) a thiol reactive moiety,    (c) —CO—NH—NH 2 ; or    (d) an enzymatic substrate or other noncovalent element of molecular recognition such as ATP, GTP, NAD, NADP, NADH, NADPH, ubiquitin, or structural analogs thereof.    
   
   
       22 . The kit of  claim 17  wherein R or L is isotopically labeled.