Methods and apparatus for analyzing biological samples by mass spectrometry

• US 6,756,586 B2
• Filed: 06/21/2002
• Issued: 06/29/2004
• Est. Priority Date: 10/15/2001
• Status: Active Grant

• Current Assignee: Vanderbilt University
• Original Assignee: Vanderbilt University
• Inventors: Caprioli, Richard
• Primary Examiner(s): Lee, John R.
• Assistant Examiner(s): Gill, Erin-Michael
• Application Number:
  US10/178,067
• Publication Number:
  US 20030073145A1
• Time in Patent Office: 739 Days
• Field of Search: 250/281, 250/282, 435/723
• US Class Current: 250/282
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

Methods and apparatuses for analyzing proteins and other biological materials and xenobiotics within a sample. A specimen is generated, which may include an energy absorbent matrix. The specimen is struck with laser beams such that the specimen releases proteins. The atomic mass of the released proteins over a range of atomic masses is measured. An atomic mass window of interest within the range of atomic masses is analyzed to determine the spatial arrangement of specific proteins within the sample, and those specific proteins are identified as a function of the spatial arrangement. By analyzing the proteins, one may monitor and classify disease within a sample.

Claims

1. A method of analyzing proteins within a sample, comprising:

(a) generating a specimen;

(b) striking the specimen with a laser beam such that the specimen releases first sample proteins;

(c) measuring the atomic mass of the released first sample proteins over a range of atomic masses;

(d) moving the specimen relative to the laser beam a predetermined linear distance;

(e) striking the specimen with the laser beam such that the specimen releases second sample proteins;

(f) measuring the atomic mass of the released second sample proteins over a range of atomic masses;

(g) analyzing an atomic mass window of interest within the range of atomic masses to determine the spatial arrangement of specific proteins within the sample; and

(h) identifying the specific proteins as a function of the spatial arrangement;

(i) comparing the specific proteins as a function of the spatial arrangement with similar data representing a normal sample; and

(j) assessing a state of disease of the sample by way of the comparing step.

2. The method as defined in claim 1, wherein the specimen comprises a tumor-bearing tissue.

3. The method as defined in claim 2, wherein the tumor-bearing tissue comprises brain tissue.

4. The method as defined in claim 2, wherein the tumor-bearing tissue comprises prostate tissue.

5. The method as defined in claim 2, wherein the tumor-bearing tissue comprises colon tissue.

6. The method as defined in claim 2, wherein the tumor-bearing tissue comprises lung tissue.

7. The method as defined in claim 2, wherein the tumor-bearing tissue comprises breast tissue.

8. The method as defined in claim 2, wherein the tumor-bearing tissue comprises rectal tissue.

9. The method as defined in claim 1, wherein generating a specimen comprises generating a fresh, frozen section.

10. The method as defined in claim 1, wherein generating a specimen comprises generating individual cells or clusters isolated by laser-capture microdissection.

11. The method as defined in claim 1, wherein analyzing the atomic mass window of interest comprises:

graphically depicting the mass of proteins within the atomic mass window of interest as a function of the linear distance.

12. The method as defined in claim 11, wherein graphically depicting the mass of proteins comprises graphically depicting in a three dimensional plot.

13. The method as defined in claim 11, further comprising:

repeating steps (b) and (c) for a plurality of laser spots on the specimen arranged within an X,Y plot; and

graphically depicting the atomic mass of proteins within the atomic mass window of interest as a function of the plurality of laser spots on the specimen arranged within the X,Y plot.

14. The method as defined in claim 1, further comprising drying the specimen prior to striking the specimen with a laser beam.

15. The method as defined in claim 1, wherein proteins within the atomic window of interest from a first laser spot are analyzed while the laser beam strikes a second laser spot.

16. The method as defined in claim 1, wherein the linear distance of movement is less than twice the width of each of successive laser spots.

17. The method as defined in claim 1, wherein identifying the specific proteins comprises extraction, HPLC fractionation, proteolysis, mass spectrometric sequencing of one or more fragments and protein database searching.

18. The method as defined in claim 1, wherein the specimen is isolated from blood.

19. The method as defined in claim 1, wherein the specimen is isolated from plasma.

20. The method as defined in claim 1, wherein the specimen comprises a breast biopsy specimen.

21. The method as defined in claim 20, wherein the specimen further comprises a scan assay chip.

22. The method as defined in claim 1, wherein the specimen comprises a tissue.

23. The method as defined in claim 22, wherein the tissue comprises brain tissue.

24. The method as defined in claim 22, wherein the tissue comprises prostate tissue.

25. The method as defined in claim 22, wherein the tissue comprises colon tissue.

26. The method as defined in claim 22, wherein the tissue comprises breast tissue.

27. The method as defined in claim 22, wherein the tissue comprises lung tissue.

28. The method as defined in claim 22, wherein the tissue comprises rectal tissue.

29. The method as defined in claim 1, wherein generating a specimen comprises generating individual cells or clusters isolated by cellular fractionating.

30. The method as defined in claim 29, wherein the cellular fractionating comprises cell sorting.

31. The method as defined in claim 30, wherein the cell sorting comprises flow cytometry.

32. The method as defined in claim 1, wherein generating a specimen comprises generating individual cells or clusters isolated by laser-capture microdissection or another cell isolation procedure.

33. A method for classifying disease, comprising:

(a) generating a diseased specimen;

(b) striking the specimen with a laser beam such that the specimen releases first sample proteins;

(c) measuring the atomic mass of the released first sample proteins over a range of atomic masses;

(d) moving the specimen relative to the laser beam a predetermined linear distance;

(e) striking the specimen with the laser beam such that the specimen releases second sample proteins;

(f) measuring the atomic mass of the released second sample proteins over a range of atomic masses;

(g) analyzing an atomic mass window of interest within the range of atomic masses to determine the spatial arrangement of specific proteins within the sample;

(h) identifying the specific proteins as a function of the spatial arrangement;

(i) correlating the identified specific proteins with one or more diseases to classify the diseased specimen;

(j) comparing the specific proteins as a function of the spatial arrangement with similar data representing a normal sample; and

(k) assessing a state of disease of the diseased specimen by way of the comparing step.

34. The method as defined in claim 33, wherein the diseased specimen comprises a tumor-bearing tissue.

35. The method as defined in claim 34, wherein the tumor-bearing tissue comprises brain tissue.

36. The method as defined in claim 34, wherein the tumor-bearing tissue comprises prostate tissue.

37. The method as defined in claim 34, wherein the tumor-bearing tissue comprises colon tissue.

38. The method as defined in claim 33, wherein generating a diseased specimen comprises generating a fresh, frozen section.

39. The method as defined in claim 33, wherein identifying the specific proteins comprises extraction, HPLC fractionation, proteolysis, mass spectrometric sequencing of one or more fragments and protein database searching.

40. A method for monitoring disease progression of a specimen over time, comprising:

(a) generating a specimen at a first time;

(b) striking the specimen with a laser beam such that the specimen releases first sample proteins;

(c) measuring the atomic mass of the released first sample proteins over a range of atomic masses;

(d) moving the specimen relative to the laser beam a predetermined linear distance;

(e) striking the specimen with the laser beam such that the specimen releases second sample proteins;

(f) measuring the atomic mass of the released second sample proteins over a range of atomic masses;

(g) analyzing an atomic mass window of interest within the range of atomic masses to determine the spatial arrangement of specific proteins within the sample;

(h) identifying the specific proteins as a function of the spatial arrangement;

(i) repeating steps (a) through (h) at a second time; and

(j) comparing the specific proteins identified at the first time with the specific proteins identified at the second time to monitor disease progression of the sample.

41. The method as defined in claim 40, further comprising correlating the identified specific proteins at the first or second times with one or more diseases.

42. The method as defined in claim 41, further comprising treating the one or more diseases.

43. The method as defined in claim 40, wherein the specimen comprises a tumor-bearing tissue.

44. The method as defined in claim 43, wherein the tumor-bearing tissue comprises brain tissue.

45. The method as defined in claim 43, wherein the tumor-bearing tissue comprises prostate tissue.

46. The method as defined in claim 43, wherein the tumor-bearing tissue comprises colon tissue.

47. The method as defined in claim 43, wherein the tumor-bearing tissue comprises lung tissue.

48. The method as defined in claim 43, wherein the tumor-bearing tissue comprises breast tissue.

49. The method as defined in claim 43, wherein the tumor-bearing tissue comprises rectal tissue.

50. The method as defined in claim 40, wherein generating a specimen comprises generating a fresh, frozen section.

51. The method as defined in claim 40, wherein identifying the specific proteins comprises extraction, HPLC fractionation, proteolysis, mass spectrometric sequencing of one or more fragments and protein database searching.

52. An apparatus for analyzing a sample containing proteins, comprising:

a laser source for sequentially striking a specimen with a laser beam at a plurality of laser spots on the specimen for sequentially releasing sample proteins from each laser spot;

a moving mechanism for sequentially moving the specimen relative to the laser beam a predetermined linear distance functionally related to the size of the laser spots prior and subsequent to the movement;

a mass analyzer for measuring the atomic mass of the released sample proteins over a range of atomic masses;

a computer for receiving atomic mass data from the mass analyzer;

a display for depicting atomic mass within an atomic mass window of interest as a function of individual laser spots on the specimen; and

a computer configured to correlate the atomic mass within the atomic mass window with one or more specific proteins and to compare the specific proteins as a function of the spatial arrangement with similar data representing a normal sample to assess a state of disease of the sample.

53. The apparatus as defined in claim 52, further comprising:

a laser mask for selectively shaping and defining sizes of laser spots on the specimen.

54. The apparatus as defined in claim 53, further comprising an optical system.

55. The apparatus as defined in claim 52, wherein atomic masses within the atomic mass window of interest are graphically depicted as a function of a plurality of laser spots on the specimen arranged within an X,Y plot.

56. A method for monitoring the development of a specimen over time, comprising:

(a) generating a specimen;

(b) striking the specimen with a laser beam such that a predetermined first laser spot on the specimen releases a first sample therapeutic agent, diagnostic agent, pharmaceutical drug or metabolite;

(c) measuring the atomic mass of the released first sample therapeutic agent, diagnostic agent, pharmaceutical drug or metabolite over a range of atomic masses;

(d) moving the specimen relative to the laser beam a predetermined linear distance related to a size of the predetermined first laser spot;

(e) striking the specimen with the laser beam such that a predetermined second laser spot on the specimen releases a second sample therapeutic agent, diagnostic agent, pharmaceutical drug or metabolite;

(f) measuring the atomic mass of the released second sample therapeutic agent, diagnostic agent, pharmaceutical drug or metabolite over a range of atomic masses;

(g) analyzing an atomic mass window of interest within the range of atomic masses to determine a spatial arrangement of specific therapeutic agent, diagnostic agent, pharmaceutical drug or metabolite within the sample;

(h) identifying the specific therapeutic agent, diagnostic agent, pharmaceutical drug or metabolite as a function of the spatial arrangement;

(i) repeating steps (a) through (h); and

(j) comparing the specific therapeutic agent, diagnostic agent, pharmaceutical drug or metabolite identified at a first time with a therapeutic agent, diagnostic agent, pharmaceutical drug or metabolite identified at a later time to monitor the development of the sample.

57. The method as defined in claim 56, further comprising correlating the identified therapeutic agent, diagnostic agent, pharmaceutical drug or metabolite with an identified protein released from the sample.