Mass spectrometric detection of polypeptides

• US 6,387,628 B1
• Filed: 09/18/2000
• Issued: 05/14/2002
• Est. Priority Date: 09/02/1997
• Status: Expired due to Term

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Little, Daniel, Köster, Hubert, Higgins, G. Scott, Lough, David
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US09/664,977
• Time in Patent Office: 603 Days
• Field of Search: 435/516, 435/91.2, 435/69.1
• US Class Current: 506/6
• 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
• CPC Class Codes: G01N 33/6851 Methods of protein analysis involving laser desorption ionisation mass spectrometry
• CPC Class Codes: H01J 49/00 Particle spectrometers or separator tubes

Abstract

A process for determining the identity of a target polypeptide using mass spectroscopy is provided. Depending on the target polypeptide to be identified, a process as disclosed can be used, for example, to diagnose a genetic disease or chromosomal abnormality, a predisposition to a disease or condition, or infection by a pathogenic organism; or for determining identity or heredity. Kits for performing the disclosed processes also are provided.

Claims

1. A method for screening for or identifying a subject having or predisposed to a disease or condition, comprising:

a) obtaining a biological sample containing a target nucleic acid from the subject and preparing a polypeptide therefrom;

b) determining the molecular mass of the polypeptide by mass spectrometry;

c) comparing the molecular mass of the polypeptide with the molecular mass of a corresponding known polypeptide, thereby determining the identity of the polypeptide, wherein;

the nucleic acid encoding the polypeptide is a marker for the disease or condition.

2. The method of claim 1, wherein the polypeptide is obtained by in vitro translation of the target nucleic acid obtained from the subject.

3. The method of claim 1, wherein the polypeptide is obtained by in vitro transcription of a nucleic acid encoding the target polypeptide and translation of RNA produced by the in vitro transcription.

4. The method of claim 1, wherein the sample is selected from the group consisting of a tissue sample, a cell sample and a biological fluid.

5. The method of claim 1, wherein the disease or condition is selected from the group consisting of Huntington'"'"'s disease, prostate cancer, Fragile X syndrome type A, myotonic dystrophy type I, Kennedy disease, Machado-Joseph disease, dentatorubral and pallidolyusian atrophy, spino bulbar muscular atrophy, and aging.

6. The method of claim 1, wherein the disease or condition is caused by an organism selected from the group consisting of a virus, a bacterium, a fungus and a protist.

7. A process for determining the amino acid sequence of a polypeptide of interest using mass spectrometry, comprising the steps of:

a) contacting the polypeptide of interest with an agent that cleaves an amino acid from a terminus of the polypeptide to produce a cleaved amino acid and a deletion fragment;

b) subjecting the cleaved amino acid or the deletion fragment to mass spectrometry; and

c) repeating step a) and step b), as necessary, thereby determining the amino acid sequence of the polypeptide.

8. The process of claim 7, wherein the polypeptide of interest is obtained by in vitro translation of an RNA encoding the polypeptide, or by in vitro transcription of a nucleic acid encoding the target polypeptide and translation of RNA produced by the in vitro transcription.

9. The process of claim 7, further comprising conditioning the polypeptide of interest prior to step a), or conditioning the cleaved amino acid or the deletion fragment prior to mass spectrometry.

10. The process of claim 9, wherein the conditioning comprises reducing the charge heterogeneity of the polypeptide, the cleaved amino acid, or the deletion fragment.

11. The process of claim 10, wherein the conditioning comprises contacting the target polypeptide with a cation exchange material.

12. The process of claim 9, wherein the conditioning comprises mass modifying the polypeptide, the cleaved amino acid, or the deletion fragment.

13. The process of claim 9, wherein the agent is a chemical agent.

14. The process of claim 7, wherein the agent is an enzyme.

15. The process of claim 14, wherein the enzyme is an aminopeptidase or a carboxypeptidase.

16. The process of claim 7, wherein the polypeptide of interest is immobilized on a solid support.

17. The process of claim 16, wherein the solid support is selected from the group consisting of a bead and a microchip.

33. A process for determining a nucleotide sequence of an unknown polynuotide using mass spectrometry, comprising the steps of:

a) determining the amino acid sequence of a polypeptide encoded by the unknown polynucleotide by mass spectrometry by the method of claim 8;

b) comparing the amino acid sequence of the unknown polypeptide to an amino acid sequence encoded by a corresponding known polynucleotide, thereby determining the nucleotide sequence of the unknown polynucleotide.

34. The process of claim 33, further comprising conditioning the polypeptide encoded by the polynucleotide prior to contacting the polypeptide with an agent that cleaves an amino acid, or conditioning the cleaved amino acid or the deletion fragment prior to mass spectrometry.

35. The process of claim 33, wherein the polypeptide encoded by the polynucleotide is immobilized to a solid support.

36. The process of claim 35, wherein the polypeptide immobilized to the solid support through a cleavable linker.

37. The process of claim 36, wherein the cleavable linker is selected from the group consisting of an acid cleavable linker and photocleavable linker.

38. The process of claim 35, wherein the immobilized polypeptides comprise an addressble array.

18. A process for determining the amino acid sequence of a polypeptide of interest using mass spectrometry, comprising the steps of:

a) producing a nested set of deletion fragments of the polypeptide; and

b) subjecting the deletion fragments to mass spectrometry, thereby determining the amino acid sequence of the polypeptide.

19. The process of claim 18, wherein the polypeptide of interest is mmobilized on a solid support prior to producing the nested set of deletion ragments.

20. The process of claim 19, wherein the polypeptide of interest is mmobilized to the solid support through a cleavable linker.

21. The process of claim 20, wherein the cleavable linker is selected from the group consisting of an acid cleavable linker and photocleavable linker.

22. A process for determining the amino acid sequence of each poly ptide in a plurality of polypeptides using mass spectroscopy, comprising the steps of:

a) differentially mass modifying each polypeptide in the plurality to produce differentially mass modified polypeptides;

b) contacting the differentially mass modified polypeptides with an agent that cleaves an amino acid from a terminus of the polypeptides to produce a cleaved amino acid and a deletion fragment;

c) subjecting the cleaved amino acid or the deletion fragment to mass spectrometry; and

d) repeating step b) and step c), as necessary, thereby determining the amino acid sequence of each polypeptide in the plurality.

23. The process of claim 22, wherein each polypeptide in the plurality is immobilized to the solid support.

24. The process of claim 23, wherein each polypeptide in the plurality is immobilized to the solid support through a cleavable linker.

25. The process of claim 24, wherein the cleavable linker is selected from the group consisting of an acid cleavable linker and photocleavable linker.

26. The process of claim 22, further comprising conditioning each polypeptide prior to step b), or conditioning the cleaved amino acid or the deletion fragment prior to mass spectrometry.

27. The process of claim 22, wherein the conditioning comprises contacting the target polypeptide with a cation exchange material.

28. The process of claim 22, wherein the agent is a chemical agent.

29. The process of claim 22, wherein the agent is an enzyme.

30. The process of claim 29, wherein the enzyme is an aminopeptidase or a carboxypeptidase.

31. The process of claim 22, wherein each polypeptide in the plurality is immobilized on a solid support.

32. The process of claim 31, wherein each polypeptide is immobilized in an array.