Method and apparatus for maldi analysis

• US 6,423,966 B2
• Filed: 01/14/2000
• Issued: 07/23/2002
• Est. Priority Date: 09/19/1996
• Status: Expired due to Term

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Hillenkamp, Franz, Reuter, Dirk, Lough, David M., Higgins, G. Scott
• Primary Examiner(s): BERMAN, JACK I
• Application Number:
  US09/483,629
• Publication Number:
  US 20020005478A1
• Time in Patent Office: 921 Days
• Field of Search: 250/288
• US Class Current: 250/288
• CPC Class Codes: B01J 2219/00387 Applications using probes
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: H01J 49/0418 for laser desorption, e.g. matrix-assisted laser desorption/ionisation [MALDI] plates or surface enhanced laser desorption/ionisation [SELDI] plates
• CPC Class Codes: H01J 49/164 Laser desorption/ionisation, e.g. matrix-assisted laser desorption/ionisation [MALDI] sample holders H01J49/0418

Abstract

Matrix assisted laser desorption/ionization is performed,in a manner to thermalize large analyte ions in a plume of desorbed material for spectroscopic analysis. The thermalized ions have a low or zero mean velocity and are presented at a well-defined instant in time, reducing artifacts and sharpening the spectral peaks. In one embodiment the light is delivered to a matrix or sample holder having a cover, baffle or compartment. The baffle or compartment impedes or contains a plume of desorbed material and the analyte undergoes collisions to lower its mean velocity and directionality. Thus “thermalized” the analyte ions are passed to a mass analysis instrument. In a preferred embodiment an optical fiber butts up against a thin transparent plate on which the specimen resides, with the matrix side in a vacuum acceleration chamber. A mechanical stage moves the specimen in both the x- and y-directions to select a point on the specimen which is to receive the radiation. The use of a fiber optic illuminator allows the entire stage assembly to be subsumed essentially within the dimensions of a conventional stage. In other embodiments, a thermalizing compartment is provided in a capillary tube about the end of the illumination fiber and the sample matrix is deposited along the inner cylindrical wall of the tube, so the capillary forms a migration path to the outlet for thermalization of the desorbed analyte. In other embodiments microstructures having the shape of a small lean-to, overhang or perforated cover plate, or providing a high aspect surface texture, provide the necessary containment to promote thermalization of the released analyte. A thin layer or cover of fibrous or permeable material may also be used to thermalize the analyte before mass analysis, and in other embodiment this material may also act as the substrate. An automated instrument may include a fixed array of illumination fibers which are illuminated at different times to eject samples from a corresponding array of points on the specimen.

Claims

1. A method, comprising:

capturing nucleic acid molecules onto a pin of a pintool;

illuminating the nucleic acid on the pin to generate desorbed nucleic acid and to partially randomize the directional momentum of the desorbed nucleic acid molecules; and

determining the molecular weight of the desorbed nucleic acid molecules by mass spectrometry.

2. The method of claim 1, wherein the pin comprises a channel.

3. The method of claim 2, wherein the pin comprises an optical fiber within the channel.

4. The method of claim 1, wherein the configuration of the tip of the pin is selected from the group consisting of flat, nanowell, concave, convex, truncated conic, and truncated pyramidal.

5. The method of claim 1, wherein the pin comprises polystyrene.

6. The method of claim 1, wherein the pin is collapsible.

7. The method of claim 1, wherein the step of capturing the nucleic acid onto the pin comprises:

contacting the pin with the nucleic acid; and

applying an electrical field to the pin, whereby the nucleic acid is captured onto the pin.

8. The method of claim 1, wherein the step of capturing the nucleic acid on the pin comprises:

contacting the pin with the nucleic acid; and

hybridizing the nucleic acid to an oligonucleotide complementary to a portion of the nucleic acid, wherein the oligonucleotide is attached to the pin, whereby the nucleic acid is captured onto the pin.

9. The method of claim 8, wherein the oligonucleotide is fully or partially single-stranded.

10. The method of claim 1, wherein:

the step of capturing the nucleic acid on the pin comprises attaching the nucleic acid to a functionalized bead; and

the functionalized bead is attached to the pin, whereby the nucleic acid is captured onto the pin.

11. The method of claim 1, further comprising the step of contacting the pin with a matrix.

12. The method of claim 11, wherein the step of contacting the pin with the matrix comprises the step of dipping the pin in the matrix.

13. The method of claim 11, wherein the step of contacting the pin with matrix comprises the step of spraying the pin with the matrix.

14. The method of claim 1, wherein the step of illuminating the nucleic acid is by top illumination.

15. The method of claim 1, wherein the step of illuminating the nucleic acid is by transmission illumination.

16. The method of claim 1, further comprising the step of contacting the pin with a wash solution.

17. The method of claim 16, wherein the wash solution comprises ammonium citrate.

18. method of claim 1, further comprising the step of amplifying the nucleic acid.

19. The method of claim 18, wherein the nucleic acid is amplified before the step of capturing the nucleic acid onto the pin.

20. The method of claim 18, wherein the nucleic acid is amplified after the step of capturing the nucleic acid onto the pin.

21. The method of claim 18, wherein the nucleic acid is amplified by a process comprising the steps of:

hybridizing a primer to the nucleic acid; and

extending the primer in the presence of deoxynucleic acids.

22. The method of claim 21, wherein the primer is attached to the tip of the pin.

23. The method of claim 1, further comprising the step of inserting the tip of the pin into a well of a microwell plate.

24. The method of claim 1, further comprising the step of contacting the pin with a probe.

25. The method of claim 24, wherein the pin is fitted through a channel in the probe.

26. The method of claim 24, wherein the probe comprises stainless steel.

27. The method of claim 24, wherein the probe comprises one channel.

28. The method of claim 24, wherein the probe comprises multiple channels.

29. The method of claim 24, wherein the probe comprises a number of channels equal to the number of pins on the pintool.

30. The method of claim 1, wherein the directional momentum of the desorbed nucleic acid is fully randomized.

31. The method of claim 1, wherein the directional momentum of the desorbed nucleic acid is at least partially randomized by thermalization.

32. The method of claim 1, wherein the directional momentum is at least partially randomized by at least partially confining the desorbed nucleic acid molecules.

33. The method of claim 1, wherein the directional momentum is at least partially randomized by two-stage ion extraction.

34. method, comprising:

hybridizing a primer nucleic acid to a sample nucleic acid;

extending the primer in the presence of at least one nucleotide to produce an extended nucleic acid, wherein the nucleotide is selected from the group consisting of deoxynucleotide and dideoxynucleotide;

capturing the extended nucleic acid onto a pin of a pintool;

illuminating the extended nucleic acid on the pin to generate desorbed nucleic acid, wherein the directional momentum of the desorbed nucleic acid is at least partially thermalized; and

determining the molecular weight of the desorbed nucleic acid by mass spectrometry.

35. The method of claim 34, further comprising the step of amplifying the sample nucleic acid prior to hybridizing the primer nucleic acid to the sample nucleic acid.

36. A pintool, comprising:

two or more pins; and

nucleic acid captured onto at least one of the pins, wherein the pins provide means for confining desorbed material to at least partially randomize the directional momentum of the desorbed material.

37. The pintool of claim 36, wherein the pin comprises a channel.

38. The pintool of claim 36, wherein the configuration of the tip of the pin is selected from the group consisting of flat, nanowell, concave, convex, truncated conic, and truncated pyramidal.

39. The pintool of claim 36, wherein the pin comprises polystyrene.

40. The pintool of claim 36, herein the pin is collapsible.

41. The pintool of claim 36, wherein the means for confining desorbed material is a probe, wherein:

the probe comprises at least one channel; and

a pin is fitted through the channel.

42. A pintool, comprising:

two or more pins;

nucleic acid captured onto at least one of the pins; and

a probe in contact with at least one of the pins, wherein;

the probe comprises at least one channel, and

a pin is fitted through the channel.

43. The pintool of claim 42, wherein the probe comprises stainless steel.

44. The pintool of claim 42, wherein the probe comprises one channel.

45. The pintool of claim 42, wherein the probe comprises multiple channels.

46. The pintool of claim 42, wherein the configuration of the tip of the pin is selected from the group consisting of flat, nanowell, concave, convex, truncated-conic, and truncated pyramidal.

47. The pintool of claim 42, wherein the pin comprises polystyrene.

48. The pintool of claim 42, wherein the pin is collapsible.

49. The pintool of claim 42, wherein the pin comprises a channel.

50. The pintool of claim 49, wherein the pin comprises an optical fiber within the channel.

51. A pintool, comprising:

two or more pins; and

nucleic acid captured onto at least one of the pins, wherein;

a pin comprises a channel; and

a pin comprises an optical fiber within a channel.

52. The pintool of claim 51, wherein the configuration of the tip of the pin is selected from the group consisting of flat, nanowell, concave, convex, truncated conic, and truncated pyramidal.

53. The pintool of claim 51, wherein the pin comprises polystyrene.

54. The pintool of claim 51, wherein the pin is collapsible.

55. A method, comprising:

hybridizing a primer nucleic acid to a sample nucleic acid;

extending the primer in the presence of at least one nucleotide to produce two or more extended nucleic acids, wherein the nucleotide is selected from the group consisting of deoxynucleotide and dideoxynucleotide;

capturing the extended nucleic acids onto a pin of a pintool;

illuminating the extended nucleic acids on the pin to generate desorbed nucleic acids and at least partially randomizing the directional momentum of the desorbed nucleic acids; and

determining the molecular weights of the nucleic acids by mass spectrometry.

56. A method, comprising:

capturing a nucleic acid onto a pin of a pintool, wherein the pintool comprises at least two pins

illuminating the nucleic acid on the pin to generate desorbed nucleic acid and to at least partially randomizing the directional momentum of the desorbed nucleic acids; and

determining the molecular weight of the desorbed nucleic acid by mass spectrometry.

57. A method comprising the steps of:

capturing a nucleic acid onto a pin of a pintool, wherein the pintool comprises at least two pins;

releasing the nucleic acid from the pin onto a substrate;

illuminating the nucleic acid on the substrate to generate desorbed nucleic acid; and

determining the molecular weight of the desorbed nucleic acid by mass spectrometry.

58. A method, comprising the steps of:

capturing a nucleic acid onto a pin of a pintool, wherein the pintool comprises at least two pins;

releasing the nucleic acid from the pin onto a substrate;

illuminating the nucleic acid on the substrate to generate desorbed nucleic acid, wherein the directional momentum of the desorbed nucleic acids is at least partially randomized; and

determining the molecular weight of the desorbed nucleic acid by mass spectrometry.