Kit for single oxygen atom incorporation into digested peptides

Abstract

Optimized enzymatic conditions incorporate a single oxygen atom into digested peptides using a peptidase. The incorporation of a single oxygen atom is especially useful for proteolytic 18 O labeling in comparative proteomics. The optimized proteolytic 18 O labeling minimizes the generation of a mixture of isotopic isoforms of the peptides resulting from incorporation of either one or two 18 O atoms. The outcome is accurate quantification of isotopically labeled peptides.

1 . A kit for incorporating a single oxygen atom into a digested peptide of a protein comprising:
 a first peptidase selected from the group consisting of peptidyl-Lys metalloendopeptidase (EC 3.4.24.20), peptidyl-Asp metalloendopeptidase (EC 3.4.24.33) and combinations thereof; and  18 O enriched water.   
     
     
         2 . The kit of  claim 1  further comprising a buffer, wherein the buffer optimizes a condition for the first peptidase. 
     
     
         3 . The kit of  claim 2 , wherein the buffer optimizes pH. 
     
     
         4 . The kit of  claim 2 , wherein the buffer optimizes salt concentration. 
     
     
         5 . The kit of  claim 1 , wherein the  18 O enriched water is about 95% H 2   18 O and about 5% H 2   16 O. 
     
     
         6 . The kit of  claim 1 , wherein a single oxygen atom is incorporated into at least about 90% of the digested peptides. 
     
     
         7 . The kit of  claim 1  further comprising a second peptidase. 
     
     
         8 . The kit of  claim 7  further comprising a buffer, wherein the buffer optimizes a condition for the second peptidase. 
     
     
         9 . The kit of  claim 7 , wherein the second peptidase is selected from the group consisting of exopeptidase (EC 3.4.11-19), endopeptidase (EC 3.4.21-25 and 99) and combinations thereof. 
     
     
         10 . The kit of  claim 9 , wherein the second peptidase is an exopeptidase selected from the group consisting of aminopeptidase (EC 3.4.11), dipeptidyl-peptidase (EC 3.4.14), tripeptidyl-peptidase (EC 3.4.14), carboxypeptidase (EC 3.4.16-18), peptidyl-dipeptidase (EC 3.4.15), dipeptidase (EC 3.4.13), omega peptidase (EC.3.4.19) and combinations thereof. 
     
     
         11 . The kit of  claim 9 , wherein the second peptidase is an endopeptidase selected from the group consisting of serine endopeptidase (EC 3.4.21), cysteine endopeptidase (EC 3.4.22), aspartic endopeptidase (EC 3.4.23), metalloendopeptidase (EC 3.4.24), threonine endopeptidase (EC 3.4.25), unassigned endopeptidase (EC 3.4.99) and combinations thereof. 
     
     
         12 . The kit of  claim 11 , wherein the second peptidase is a metalloendopeptidase selected from the group consisting of peptidyl-Lys metallopeptidase (EC 3.4.24.20), peptidyl-Asp metalloendopeptidase (EC 3.4.24.33), thermolysin (EC 3.4.24.27), mycolysin (EC 3.4.24.31) and combinations thereof.