IR MALDI MASS SPECTROMETRY OF NUCLEIC ACIDS USING LIQUID MATRICES

• US 20010055811A1
• Filed: 05/07/1998
• Published: 12/27/2001
• Est. Priority Date: 05/07/1998
• Status: Active Grant

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Franz Hillenkamp
• Inventors: HILLENKAMP, FRANZ
• Granted Patent: US 6,723,564 B2
• Time in Patent Office: Days
• US Class Current: 436/43
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: H01J 49/164 Laser desorption/ionisation, e.g. matrix-assisted laser desorption/ionisation [MALDI] sample holders H01J49/0418
• CPC Class Codes: Y10T 436/11 Automated chemical analysis
• CPC Class Codes: Y10T 436/14 Heterocyclic carbon compound [i.e. , O, S, N, Se, Te, as only ring hetero atom]
• CPC Class Codes: Y10T 436/142222 Hetero-O [e.g., ascorbic acid, etc.]
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry
• CPC Class Codes: Y10T 436/25 including sample preparation
• CPC Class Codes: Y10T 436/25875 Gaseous sample or with change of physical state

Abstract

Mass spectrometry of large nucleic acids by infrared Matrix-Assisted Laser Desorption/Ionization (MALDI) using a liquid matrix is reported.

Claims

1. A process for performing matrix assisted laser desorption/ionization (MALDI) of a nucleic acid in preparation for analysis by mass spectrometry, comprising the steps of:

(a) depositing a solution containing the nucleic acid and a liquid matrix on a substrate, thereby forming a homogeneous, thin layer of a nucleic acid/liquid matrix solution; and

(b) illuminating the substrate with infrared radiation, so that the nucleic acid in the solution is desorbed and ionized.

2. A process of

claim 1

, wherein the liquid matrix has at least one of the following desirable properties:

i) is miscible with a nucleic acid compatible solvent, ii) is vacuum stable, and iii) is of an appropriate viscosity to facilitate dispensing of micro- to nano-liter volumes of matrix alone or mixed with a nucleic acid compatible solvent.

3. A process of

claim 1

, wherein the liquid matrix contains at least one functional group that strongly absorbs infrared radiation.

4. A process of

claim 3

, wherein the functional group is selected from the group comprising:

nitro, sulfonyl, sulfonic acid, sulfonamide, nitrile, carbonyl, aldehyde, carboxylic acid, amide, ester, anhydride, ketone, amine, hydroxyl, an aromatic ring and a diene.

5. A process of

claim 1

, wherein the liquid matrix is selected from a group comprising:

an alcohol, a carboxylic acid, a primary or secondary amide, a primary or secondary amine, a nitrile, hydrazine and hydrazide.

6. A process of

claim 5

, wherein the alcohol is selected from the group comprising:

glycerol, 1,2- or 1,3- propane diol, 1,2-, 1,3- or 1,4- butane diol and triethanolamine.

7. A process of

claim 5

, wherein the carboxylic acid is selected from the group comprising:

lactic acid, acetic acid, formic acid, propionic acid, butanoic acid, pentanoic acid, hexanoic acid and esters thereof.

8. A process of

claim 5

, wherein the amide is selected from the group comprising:

acetamide, propanamide, butanamide, pentanamide and hexanamide, whether branched or unbranched.

9. A process of

claim 5

, wherein the amine is selected from the group comprising:

propylamine, butylamine, pentylamine, hexylamine, heptylamine, diethylamine and dipropylamine.

10. A process of

claim 2

, wherein the liquid matrix is comprised of at least two liquids, which confer at least one of the desirable properties.

11. A process of

claim 1

, wherein the liquid matrix comprises an additive.

12. A process of

claim 11

, wherein the additive is selected from the group comprising:

a compound having a high extinction coefficient at the laser wavelength used for the analysis, an additive that acidifies the liquid matrix, an additive that minimizes salt formation between the liquid matrix and the phosphate backbone of the nucleic acid.

13. A process of

claim 5

, wherein prior to step (a), the liquid matrix is treated to minimize salt formation between the matrix and the phosphate backbone of the nucleic acid.

14. A process of

claim 13

, wherein the liquid matrix is treated by distillation or ion exchange.

15. A process of

claim 1

, wherein the liquid matrix is selected from the group consisting of:

glycerol, lactic acid or triethanolamine.

16. A process of

claim 15

, wherein the liquid matrix is glycerol and the final analyte-to-glycerol molar ratio is about 10⁻

4 to about 10<sup>−

9</sup> depending on the mass of the nucleic acid and the total sample volume.

17. A process of

claim 1

, wherein the liquid matrix is glycerol, the mass of the nucleic acid is in the range of from about 10⁴ to about 10⁶ Da and the glycerol is subjected to ion exchange prior to step (a).

18. A process of

claim 1

, wherein the nucleic acid is DNA.

19. A process of

claim 18

, wherein the DNA is less than or equal to about a 2000-mer.

20. A process of

claim 1

, wherein the nucleic acid is RNA.

21. A process of

claim 20

, wherein the RNA is less than or equal to about a 1200-mer.

22. A process of

claim 1

, wherein the infrared radiation is of a wavelength in the range of from about 2.5 μ

m to about 12 μ

m.

23. A process of

claim 1

, wherein the radiation pulses have a width in the range of about 500 ps to about 500 ns.

24. A process of

claim 1

, wherein the infrared radiation is generated from a source selected from the group comprising:

a CO laser, a CO₂ laser, an Er laser and an optical parametric oscillator laser emitting in the range of about 2.5 to about 12 μ

m.

25. A process of

claim 1

, wherein the sample contains less than about 10 pmoles of nucleic acid.

26. A process of

claim 1

, wherein step (a) is performed using an automated device.

27. A process of

claim 1

, wherein the sample is cooled to a temperature, which is below about 20°

C.

28. A process of

claim 1

, wherein the sample is heated to a temperature which is greater than about 20°

C. and less than about 80°

C.

29. A process for analyzing a nucleic acid by mass spectrometry, comprising the steps of:

(a) depositing a solution containing the nucleic acid and a liquid matrix on a substrate, thereby forming a homogeneous, thin layer of a nucleic acid/liquid matrix solution;

(b) illuminating the substrate of (a) with an infrared laser, so that the nucleic acid is desorbed and ionized; and

(c) mass separating and detecting the ionized nucleic acid using an appropriate mass separation and analysis format.

30. A process of

claim 29

, wherein the liquid matrix has at least one of the following desirable properties:

i) is miscible with a nucleic acid compatible solvent, ii) is vacuum stable, and iii) is of an appropriate viscosity to facilitate dispensing of micro- to nano-liter volumes of matrix alone or mixed with a nucleic acid compatible solvent.

31. A process of

claim 29

, wherein the liquid matrix contains at least one functional group that strongly absorbs infrared radiation.

32. A process of

claim 31

, wherein the functional group is selected from the group comprising:

nitro, sulfonyl, sulfonic acid, sulfonamide, nitrile, carbonyl, aldehyde, carboxylic acid, amide, ester, anhydride, ketone, amine, hydroxyl, an aromatic ring and a diene.

33. A process of

claim 29

, wherein the liquid matrix is selected from a group comprising:

an alcohol, a carboxylic acid, a primary or secondary amide, a primary or secondary amine, a nitrile, hydrazine and hydrazide.

34. A process of

claim 33

, wherein the alcohol is selected from the group comprising:

glycerol, 1,2- or 1,3-propane diol, 1,2-, 1,3- or 1,4-butane diol and triethanolamine.

35. A process of

claim 33

, wherein the carboxylic acid is selected from the group comprising:

lactic acid, acetic acid, formic acid, propionic acid, butanoic acid, pentanoic acid, hexanoic acid and esters thereof.

36. A process of

claim 33

, wherein the amide is selected from the group comprising:

acetamide, propanamide, butanamide, pentanamide and hexanamide, whether branched or unbranched.

37. A process of

claim 33

, wherein the amine is selected from the group comprising:

propylamine, butylamine, pentylamine, hexylamine, heptylamine, diethylamine and dipropylamine.

38. A process of

claim 30

, wherein the liquid matrix is comprised of at least two liquids, which confer at least one of the desirable properties.

39. A process of

claim 29

, wherein the liquid matrix comprises an additive.

40. A process of

claim 39

, wherein the additive is selected from the group comprising:

a compound having a high extinction coefficient at the laser wavelength used for the analysis, an additive that acidifies the liquid matrix, an additive that minimizes salt formation between the liquid matrix and the phosphate backbone of the nucleic acid.

41. A process of

claim 33

, wherein prior to step (a), the liquid matrix is treated to minimize salt formation between the matrix and the phosphate backbone of the nucleic acid.

42. A process of

claim 41

, wherein the liquid matrix is treated by distillation or ion exchange.

43. A process of

claim 29

, wherein the liquid matrix is selected from the group consisting of:

glycerol, lactic acid or triethanolamine.

44. A process of

claim 43

, wherein the liquid matrix is glycerol and the final analyte-to-glycerol molar ratio is about 10⁻

4 to about 10<sup>−

9</sup> depending on the mass of the nucleic acid and the total sample volume.

45. A process of

claim 29

, wherein the liquid matrix is glycerol, the mass of the nucleic acid is in the range of from about 10⁴ to about 10⁶ Da and the glycerol is subjected to ion exchange prior to step (a).

46. A process of

claim 29

, wherein the nucleic acid is DNA.

47. A process of

claim 46

, wherein the DNA is less than or equal to about a 2000-mer.

48. A process of

claim 29

, wherein the nucleic acid is RNA.

49. A process of

claim 48

, wherein the RNA is less than or equal to about a 1200-mer.

50. A process of

claim 29

, wherein the infrared radiation is of a wavelength in the range of from about 2.5 μ

m to about 12 μ

m.

51. A process of

claim 29

, wherein the radiation pulses have a width in the range of about 500 ps to about 500 ns.

52. A process of

claim 29

, wherein the infrared radiation is generated from a source selected from the group comprising:

a CO laser, a CO₂ laser, an Er laser and an optical parametric oscillator laser emitting in the range of about 2.5 to about 12 μ

m.

53. A process of

claim 29

, wherein the sample contains less than about 10 pmoles of nucleic acid.

54. A process of

claim 29

, wherein step (a) is performed using an automated device.

55. A process of

claim 29

, wherein the sample is cooled to a temperature, which is below about 20°

C.

56. A process of

claim 29

, wherein the sample is heated to a temperature which is greater than about 20°

C. and less than about 80°

C.

57. A method of

claim 29

, wherein prior to step (c), the nucleic acid ions are extracted from the ion source by delayed extraction.

58. A method of

claim 29

, wherein the mass separation and analysis format is selected from the group consisting of:

time-of-flight (TOF), quadrupole, magnetic sector, Fourier transform ion cyclotron resonance (FTICR), ion trap or a combination thereof.

59. A method of

claim 58

, wherein the TOF is linear.

60. A method of

claim 58

, wherein the TOF has a reflector.

61. A method of

claim 58

, wherein the TOF reflector has a linear field.

62. A method of

claim 58

, wherein the TOF reflector has a nonlinear field.

63. A method of

claim 58

, wherein the quadrupole is single.

64. A method of

claim 58

, wherein the quadrupole is multiple.

65. A method of

claim 58

, wherein the magnetic sector is single.

66. A method of

claim 58

, wherein the magnetic sector is multiple.