Method for single oxygen atom incorporation into digested peptides using peptidases
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 method for the comparison of a protein under different biological conditions using mass spectrometry comprising the steps of:
a. digesting said protein of a first biological condition into peptides with either a peptidyl-Lys metalloendopeptidase or a peptidyl-Asp metalloendopeptidase in a buffer using 18 O enriched water to incorporate a single 18 O atom into at least 90% of the digested peptides;
b. digesting said protein of a second biological condition into peptides with said peptidyl-Lys metalloendopeptidase or peptidyl-Asp metalloendopeptidase in the buffer using 16 O water to incorporate a single 16 O atom into the digested peptides;
c. mixing digested peptides containing enriched 18 O of the first biological condition and digested peptides containing 16 O of the second biological condition;
d. performing mass spectrometry analysis of the relative ratio of 18 O and 16 O in said mixed digested peptides; and
e. determining a ratio of labeled peptides to unlabeled peptides, wherein the ratio determined indicates a change in an amount of said protein of a second biological condition relative to an amount of said protein of a first biological condition.
2. The method of claim 1 , wherein said comparison is used for the identification of a biological condition in an animal.
3. The method of claim 1 , wherein said 18 O enriched water is 95% H 2 18 O and 5% H 2 16 O.
4. The method of claim 1 , wherein the protein digest of step (a) and the protein digest of step (b) further comprise digesting said protein with another peptidase which is an exopeptidase (EC 3.4.11-19) or an endopeptidase (EC 3.4.21-25 and 99).
5. The method of claim 4 , wherein said exopeptidase is an aminopeptidase (EC 3.4.11), a dipeptidyl-peptidase EC 3.4.14), tripeptidyl-peptidase (EC 3.4.14), a carboxypeptidase (EC 3.4.16-18), a peptidyl-dipeptidase (EC 3.4.15), a dipeptidase (EC 3.4.13) or an omega peptidase (EC 3.4.19).
6. The method of claim 4 , wherein said endopeptidase is a serine endopeptidase (EC 3.4.21), a cysteine endopeptidase (EC 3.4.22), an aspartic endopeptidase (EC 3.4.23), a metalloendopeptidase (EC 3.4.24) a threonine endopeptidase (EC 3.4.25) or an unassigned endopeptidase (EC 3.4.99).
7. The method of claim 6 , wherein said metalloendopeptidase is thermolysin (EC 3.4.24.27) of mycolysin (EC 3.4.24.31).
8. The method of claim 1 , wherein digesting said protein of the first biological condition comprises:
denaturing said protein;
blocking a thiol group; and
digesting said protein with a peptidase in said buffer using 18 O enriched water.