Isotope distribution encoded tags for protein identification

• US 6,629,040 B1
• Filed: 03/20/2000
• Issued: 09/30/2003
• Est. Priority Date: 03/19/1999
• Status: Expired due to Fees

• Current Assignee: Battelle Memorial Institute, University of Washington
• Original Assignee: Battelle Memorial Institute, University of Washington
• Inventors: Bruce, James E., Rist, Beate, Smith, Richard D., Goodlett, David R., Aebersold, Ruedi
• Primary Examiner(s): Brusca, John S.
• Assistant Examiner(s): Zhou, Shubo
• Application Number:
  US09/528,603
• Time in Patent Office: 1,289 Days
• Field of Search: 702/19, 702/23, 250/281, 530/300.35
• US Class Current: 702/23
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2458/15 Non-radioactive isotope labels, e.g. for detection by mass spectrometry
• CPC Class Codes: G01N 33/534 with radioactive label
• CPC Class Codes: G01N 33/6815 containing sulfur, e.g. cysteine, cystine, methionine, homocysteine
• CPC Class Codes: G01N 33/6818 Sequencing of polypeptides
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry

Abstract

The present invention provides methods and reagents for identifying specific masses of compounds in a complex mixture. In particular, the present invention describes an isotope distribution encoded tag (IDEnT) concept wherein the ability to distinguish an analyte encoded with a non-native isotope from those not encoded with the isotope or from those encoded with a different isotope is used for protein identification, enzyme active site identification, peptide sequencing, and the like. Reagents used in the IDEnT strategy consist of a chemical element with a unique isotope distribution not normally found in the analyte class being studied, chemically bonded to, or an integral part of a chemical reagent with a high selectivity of affinity for specific functional group in the analyte.

Claims

1. A method for identifying a peptide or protein in a mixture comprising one or more peptides or proteins which comprises the steps of:

(a) labeling one or more peptides or proteins in the mixture with a first isotope-distribution tag reagent which comprises an element having an isotope distribution not normally found in the one or more peptides or proteins in the mixture and which selectively reacts and bonds to a functional group in one or more peptides or proteins in the mixture to give one or more labeled peptides or proteins;

(b) digesting the labeled peptides or proteins in the mixture using a digestion method that is selected to generate one or more digested peptides or proteins from the labeled peptides or proteins in the mixture that are selectively labeled with the first isotope-distribution tag reagent;

(c) measuring the masses of the digested peptides or proteins selectively labeled with the first isotope-distribution tag reagent employing mass spectrometry to give the masses of digested peptides or proteins selectively labeled with the first isotope-distribution tag reagent; and

(d) comparing the mass of one and only one single labeled digested peptide or protein determined in step (c) with one or more databases comprising the masses of peptides or proteins generated by digestion of known peptides or proteins using the digestion method of step (b) to identify the peptide or protein in the database from which the single digested peptide or protein is derived and to thereby identify one of the peptides or proteins in the mixture.

2. The method of claim 1 wherein the digestion method is digestion with an endopeptidase.

3. The method of claim 2 wherein the digestion method is digestion with trypsin.

4. The method of claim 1 wherein the isotope-distribution tag reagent selectively reacts with cysteine to label cysteines in one or more peptides or proteins in the mixture with an isotope distribution not normally found in the one or more peptides or proteins in the mixture.

5. The method of claim 1 wherein the isotope-distribution tag reagent selectively reacts with tryptophan to label tryptophan in one or more peptides or proteins in the mixture with an isotope distribution not normally found in the one or more peptides or proteins of the mixture.

6. The method of claim 1 wherein the isotope-distribution tag reagent selectively reacts with methionine to label methionine in one or more peptides or proteins in the mixture with an isotope distribution not normally found in the one or more peptides or proteins of the mixture.

7. The method of claim 1 wherein, in comparing the measured mass of the single digested labeled peptide or protein to those in the database, the presence of the functional group in a labeled peptide is used to exclude peptides in the database from the mass comparison.

8. The method of claim 7 wherein in comparing the measured mass of the single digested labeled peptide or protein to those in the database, the measured or estimated molecular weight of the protein or proteins to be identified is used to exclude peptides in-the database from the mass comparison.

9. The method of claim 1 further comprising labeling one or more peptides or proteins in the mixture with a second isotope-distribution tag reagent which comprises an element having an isotope distribution not normally found in the one or more peptides or proteins of the mixture and different from the element in the first isotope-distribution tag reagent and which selectively reacts with a functional group different from the functional group with which the first isotope-distribution tag reagent reacts.

10. The method of claim 9 wherein the first and second isotope distribution tag reagents are used to tag either end of the one or more peptides or proteins in the mixture.

11. The method of claim 10 wherein the first isotope-distribution tag reagent labels amino termini and the second isotope-distribution tag reagent labels carboxy termini.

12. The method of claim 9 wherein the first and second isotope-distribution tag reagents contain a different number of chlorine atoms.

13. The method of claim 1 wherein the first isotope-distribution tag reagent comprises one or more chlorine atoms.

14. The method of claim 1 wherein the one or more peptides or proteins in the mixture are separated by size, molecular weight or on an affinity column prior to step (a).

15. The method of claim 14 wherein steps (b)-(d) are conducted on separate samples of a given protein mixture employing more than one digestion method.

16. The method of claim 15 wherein the protein is an enzyme and the isotope-distribution tag reagent is an inhibitor of the enzyme.

17. The method of claim 1 wherein the one or more digested peptides or proteins are separated by size, molecular weight or on an affinity column prior to step (c).

18. The method of claim 1 further comprising the step of determining the amino acid sequence of one or more labeled peptides or proteins using collision-induced dissociation mass spectrometry.

19. The method of claim 1 wherein the measurement of the masses of the one or more digested peptides or proteins of step (c) is performed by Fourier Transform Ion Cyclotron-Mass Spectrometry.

20. The method of claim 1 where the first isotope-distribution tag reagent comprises two substantially chemically identical compounds which differ from each other by the substitution of a heavy isotope for at least one atom.

21. The method of claim 20 wherein the heavy isotope is deuterium which is substituted for hydrogen in one of the two reagents.

22. The method of claim 20 wherein the heavy isotope is ¹³C which is substituted for ¹²C in one of the two reagents.

23. The method of claim 1 wherein the isotope-distribution tag reagent binds to an active site or a ligand binding site in the one or more peptides or proteins in the mixture.

24. The method of claim 23 wherein an enzyme is labeled with an isotope distribution tag reagent that is an inhibitor of said enzyme.

25. The method of claim 1 further comprising the step of chemically reducing disulfide bonds in the one or more peptides or proteins in the mixture and wherein the isotope-distribution tag reagent bonds to a free sulihydryl group.

26. The method of claim 1 wherein the one or more peptides or proteins in the mixture are digested using cyanogen bromide.

27. The method of claim 1 which is applied to the identification of proteins in a mixture of proteins.

28. The method of claim 1 wherein the isotope-distribution tag reagent is a mixture of acetyl chloride in methanol and deuterated methanol.

29. The method of claim 1 wherein the mixture of peptides and proteins contains phosphoproteins and the isotope-distribution tag reagent bonds to phosphate groups.

30. The method of claim 29 wherein the isotope-distribution tag reagent comprises metal ions with a negative mass defect which have an affinity for phosphate.

31. The method of claim 1 wherein the isotope-distribution tag reagent alkylates cysteine and contains a biotin functional group.

32. The method of claim 1 wherein the isotope-distribution tag reagent is a protein crosslinking reagent.

33. The method of claim 1 wherein the isotope distribution tag reagent has the formula:

where n is an integer which can be 1-5.

34. The method of claim 33 wherein in the isotope-distribution tag reagent n is 1 and the phenyl ring is substituted with 1-4 chlorines.

35. The method of claim 1 wherein the step of measuring the masses of the one or more digested peptides or proteins is performed using an FT-ICR mass spectrometer.

36. The method of claim 1 wherein the step of measuring the masses of the one or more digested peptides or proteins is performed using a mass spectrometer having a resolution of about 5000 to about 10,000@ 1000μ

.

37. The method of claim 1 wherein the step of measuring the masses of the one or more digested peptides or proteins is performed using a mass spectrometer having a resolution of about 10,000@ 1000μ

or higher.

38. The method of claim 1 wherein the step of measuring the masses of the one or more digested peptides or proteins is performed using a mass spectrometer having an accuracy of about 10 to about 2.0 ppm at 1000μ

.

39. The method of claim 1 wherein the step of measuring the masses of the one or more digested peptides or proteins is performed using a mass spectrometer having an accuracy of about 2.0 ppm at 1000μ

or higher.

40. A method for identifying a protein in a mixture of proteins that is at least in part encoded by a known expressed sequence tag which comprises the steps of:

(a) labeling a protein in the mixture with a first isotope-distribution tag reagent which comprises an element having an isotope distribution not normally found in the proteins of the mixture and which selectively reacts and bonds to a functional group in a protein to give a labeled protein;

(b) digesting the labeled protein using a digestion method that is selective for a cut site in the protein to generate digested peptides including peptides that are selectively labeled with the first isotope-distribution tag reagent derived from proteins in the labeled protein mixture;

(c) measuring the masses of the digested peptides selectively labeled with the first isotope-distribution tag reagent employing mass spectrometry to give the masses of the digested labeled peptides; and

(d) comparing the mass of one and only one single digested labeled peptide determined in step (c) with one or more databases comprising the masses of peptides encoded by known expressed sequence tags to identify peptides in the database that correspond to the single digested peptide and thereby determine which protein(s) in the mixture are encoded at least in part by a given expressed sequence tag.

41. A method for identifying a peptide or protein in a mixture comprising the steps:

(a) labeling one or more peptides or proteins in a mixture with an isotope-distribution tag reagent that selectively labels one or more amino acids in one or more peptides or proteins in the mixture to give one or more labeled peptides or proteins;

(b) determining the masses of the labeled peptides or proteins by mass-spectrometry to give the masses of the peptides or proteins; and

(c) identifying a peptide or protein in a database using the mass of one and only one single labeled peptide or protein and a sequence constraint derived from the specificity of the first isotope-distribution tag reagent;

wherein the mass of the single peptide or protein and the sequence constraint identifies a peptide or protein in the database.

42. The method of claim 41 further comprising digesting the mixture using a digestion method that is selected to generate one or more digested peptides or proteins.

43. The method of claim 42 wherein step (c) is performed without determining the sequence of any ion derived from said one or more labeled peptides or proteins.

44. The method of claim 41 wherein step (c) is performed without -determining the sequence of any ion derived from said one or more labeled peptides or proteins.

45. A method for identifying a peptide or protein in a mixture comprising the steps:

(a) labeling one or more peptides or proteins in a mixture with an isotope-distribution tag reagent that selectively labels one or-more uncommon amino acids in one or more peptides or proteins in the mixture to give one or more labeled peptides or proteins;

(b) digesting the mixture using a digestion method that is selected to generate one or more digested labeled peptides or proteins;

(c) determining the masses of said digested labeled peptides or proteins by mass-spectrometry to give the masses of the digested labeled peptides or proteins; and

(d) identifying a protein or peptide in a database using the mass of one and only one single digested labeled peptide or protein and a sequence constraint derived from the specificity of the first isotope-distribution tag reagent;

wherein the mass of the single digested labeled peptide or protein and the sequence constraint identifies a peptide or protein in the database.

46. The method for identifying a peptide or protein in a mixture according to claim 45 wherein the isotope-distribution tag reagent selectively labels an uncommon amino acid selected from cysteine, methionine, or tryptophan.

47. A method for identifying a peptide or protein in a mixture comprising the steps:

(a) labeling one or more peptides or proteins in a mixture with an isotope-distribution tag reagent that selectively labels one or more amino acids in one or more peptides or proteins in the mixture to give one or more labeled peptides or proteins;

(b) fragmenting the peptides or proteins in the mixture in a mass spectrometer to generate one or more fragmented peptides or proteins selectively labeled with an isotope-distribution tag reagent;

(c) determining the masses of said fragmented peptides or proteins by mass-spectrometry to give the masses of one or more fragmented peptides or proteins selectively labeled with an isotope-distribution tag reagent; and

(d) identifying a protein or peptide in a database using the mass of one and only one single fragmented peptide or protein selectively labeled with an isotope-distribution tag reagent and a sequence constraint derived from the specificity of the first isotope-distribution tag reagent and without determining the sequence of any ion derived from the one or more digested labeled peptides or proteins;

wherein the mass of the single fragmented peptide or protein and the sequence constraint identifies a peptide or protein in the database.