Multiplex spatial profiling of gene expression

• US 7,569,392 B2
• Filed: 01/07/2005
• Issued: 08/04/2009
• Est. Priority Date: 01/08/2004
• Status: Active Grant

• Current Assignee: Vanderbilt University
• Original Assignee: Vanderbilt University
• Inventors: Levy, Shawn, Caprioli, Richard M.
• Primary Examiner(s): Martinell; James
• Application Number:
  US11/031,973
• Publication Number:
  US 20050196786A1
• Time in Patent Office: 1,670 Days
• Field of Search: None
• US Class Current: 436/6
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2523/319 Photocleavage, photolysis, photoactivation
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2563/167 Mass label
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: G01N 2458/15 Non-radioactive isotope labels, e.g. for detection by mass spectrometry
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry

Abstract

The present invention provides mass tag complexes that permit simultaneously obtaining information of a plurality of biological molecules. The biological molecules may be RNA or protein, and the information includes both level of expression as well as spatial disposition within a cell or tissue. The mass tag comprise a core structure, a target binding structure (e.g., nucleic acid or peptide binding structure), a cleavable linker and a mass tag that exhibits a unique mass spectroscopy signal.

Claims

1. A mass tag complex comprising:

(i) a water-soluble core structure;

(ii) a target binding agent;

(iii) a mass unit that permits detection by mass spectroscopy; and

(iv) a cleavage site connected to said mass unit.

2. The complex of claim 1, further comprising a spacer unit connected to said cleavage site and/or said target binding agent.

3. The complex of claim 1, wherein said cleavage site is susceptible to photocleavage, chemical cleavage or enzymatic cleavage.

4. The complex of claim 1, wherein said mass unit is a peptide.

5. The complex of claim 1, wherein said target binding agent is an oligonucleotide that hybridizes to an RNA of interest.

6. The complex of claim 5, wherein said oligonucleotide is about 8 to about 25 bases in length.

7. The complex of claim 1, wherein said target binding agent is an antibody that binds to a protein of interest.

8. The complex of claim 7, wherein said antibody is Ig, F(ab), F(ab′

)₂ or single chain.

9. The complex of claim 1, wherein said core structure is a polar-neutral, water-soluble structure.

10. The complex of claim 1, wherein each of said target binding agent and said mass unit is independently linked to said core structure.

11. A population of mass tag complexes, each complex comprising:

(i) an identical core structure;

(ii) a target binding agent, wherein said population comprises a plurality of target binding agents with different target binding specificities;

(iii) a mass unit that permits detection by mass spectroscopy, wherein said population comprises a plurality of mass units that have different masses that can be differentiated by said mass spectroscopy; and

(iv) a cleavage site connected to said mass unit.

12. The population of claim 11, wherein each complex further comprises a spacer unit connected to said each of cleavage sites and/or said target binding agents.

13. The population of claim 11, wherein each of said mass units is a peptide.

14. The population of claim 11, wherein each of said cleavage sites is photocleaved, enzymatically cleaved or chemically cleaved.

15. The population of claim 11, wherein said each of said target binding agents is an oligonucleotide that hybridizes to a different RNA of interest.

16. The population of claim 15, wherein each of said oligonucleotides is about 8 to about 25 bases in length.

17. The population of claim 11, wherein each of said target binding agents is an antibody that binds to a different protein of interest.

18. The population of claim 17, wherein each of said antibodies is Ig, F(ab), F(ab′

)₂ or single chain.

19. The population of claim 11, wherein each of said core structures is a polar-neutral, water-soluble structure.

20. The population of claim 11, wherein each of said target binding agents and said mass units is independently linked to said core structure.

21. A method of simultaneously obtaining information on a plurality of distinct biomolecules comprising:

(a) providing a population of mass tag complexes, each complex comprising;

(i) an identical core structure;

(ii) a target binding agent, wherein said population comprises a plurality of binding agents with different target binding specificities;

(iii) a mass unit that permits detection by mass spectroscopy, wherein said population comprises a plurality of mass units that have different masses that can be differentiated by said mass spectroscopy; and

(iv) a cleavage site connected to said mass unit;

(b) contacting said population with a biomolecule-containing sample;

(c) cleaving said cleavage site; and

(d) subjecting said sample to mass spectroscopy.

22. The method of claim 21, wherein said mass spectroscopy is MALDI-TOF.

23. The method of claim 21, wherein cleavage site is photocleaved, and step (c) comprises subjecting said sample to an appropriate light source.

24. The method of claim 21, further comprising the step, between steps (b) and (c), of spatially fixing said biomolecules and target binding agents.

25. The method of claim 24, wherein said biomolecules and target binding agents are located in a cell.

26. The method of claim 25, wherein said cell is from a patient with a pathologic condition.

27. The method of claim 26, wherein said pathologic condition is cancer, an inflammatory disease, an infection, or a developmental disease.

28. The method of claim 25, wherein said cell or patient has been treated with a therapy.

29. The method of claim 25, wherein said cell is in comprised within an intact tissue specimen or an organism.

30. The method of claim 21, wherein said mass unit is a peptide.

31. The method of claim 21, wherein each of said target binding agents is an oligonucleotide that hybridizes to a different RNA of interest.

32. The method of claim 31, wherein said oligonucleotide is about 8 to about 25 bases in length.

33. The method of claim 21, wherein each of said target binding agents is an antibody that binds to a different protein of interest.

34. The method of claim 33, wherein said antibody is Ig, F(ab), F(ab′

)₂ or single chain.

35. The method of claim 21, wherein said core structure is a polar-neutral, water-soluble structure.

36. A method of obtaining information on the spatial position of a biomolecule in a cell comprising:

(a) providing a mass tag complex comprising;

(i) a core structure;

(ii) a target binding agent;

(iii) a mass unit that permits detection by mass spectroscopy, wherein said population comprises a plurality of mass units that have different masses that can be differentiated by said mass spectroscopy; and

(iv) a cleavage site connected to said mass unit;

(b) contacting said mass tag complex with a cell-containing sample;

(c) spatially fixing said biomolecule and target binding agent;

(d) cleaving said cleavage site; and

(e) subjecting said sample to mass spectroscopy.

37. The method of claim 36, further comprising obtaining information on the expression level of said biomolecule.

38. The method of claim 36, further comprising obtaining information on a plurality of biomolecules by providing a population of mass tag complexes, each complex comprising an identical core structure;

a target binding agent with a distinct target specificity;

a distinct mass unit that permits detection by mass spectroscopy; and

a cleavage site connected to said mass unit.

39. The method of claim 38, wherein said biomolecule is an RNA or a protein.

40. The method of claim 36, wherein said cell is located in an intact tissue specimen or an organism.