IR MALDI mass spectrometry of nucleic acids using liquid matrices

• US 6,723,564 B2
• Filed: 05/07/1998
• Issued: 04/20/2004
• Est. Priority Date: 05/07/1998
• Status: Expired due to Fees

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Hillenkamp, Franz
• Primary Examiner(s): Soderquist, Arlen
• Application Number:
  US09/074,936
• Publication Number:
  US 20010055811A1
• Time in Patent Office: 2,175 Days
• Field of Search: 436/43, 436/91, 436/93-94, 436/86, 436/173, 436/174, 436/181
• US Class Current: 436/94
• 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 and analysis by mass spectrometry, comprising the steps of:

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

the liquid matrix comprises glycerol, 1,2- or 1,3- propane diol, 1,2-, 1,3-or 1,4- butane diol and triethanolamine;

(b) illuminating the substrate with infrared radiation, whereby the nucleic acid in the solution is desorbed and ionized; and

(c) determining the mass of the nucleic acid by a mass spectrometric format for separation and detection of desorbed and ionized molecules, wherein the mass of the nucleic acid molecule is at least about 90 kD and the accuracy for determining the mass of the nucleic acid is within about 1%.

2. A process of claim 1, wherein:

the liquid matrix is comprised of the alcohol and an additional liquid, which confers to the liquid matrix at least one of the properties selected from the group consisting of i) miscibility with a nucleic acid compatible solvent, ii) vacuum stability, and, iii) 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 comprises an additive.

4. A process of claim 3, wherein the additive is selected from the group consisting of a compound having a high extinction coefficient at the laser wavelength used for the analysis, an additive that acidifies the liquid matrix, and an additive that minimizes salt formation between the liquid matrix and the phosphate backbone of the nucleic acid.

5. A process of claim 1, 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.

6. A process of claim 5, wherein the liquid matrix is treated by distillation or ion exchange.

7. A process of claim 1, wherein the liquid matrix is glycerol.

8. A process of claim 7, wherein 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.

9. 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 10⁶Da and the glycerol is subjected to ion exchange prior to step (a).

10. A process of claim 1, wherein the nucleic acid is DNA.

11. A process of claim 10, the DNA is less than or equal to about a 2000-mer.

12. A process of claim 1, wherein the nucleic acid is RNA.

13. A process f claim 12, wherein the RNA is less than or equal to about a 1200-mer.

14. 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.

15. A process of claim 1, wherein the radiation pulses have a width in the range of about 500 ps to about 500 ns.

16. A process of claim 1, wherein the infrared radiation is generated from a source selected from the group consisting of a CO laser, a CO₂ laser, en Er laser and an optical parametric oscillator laser emitting in the range of about 2.5 to about 12 μ

m.

17. A process of claim 1, wherein the sample contains less than about 10 pmoles of nucleic acid.

18. A process of claim 1, wherein step (a) is automated.

19. A process of claim 1, wherein the sample, which with the matrix remains in a liquid or glass state, is cooled to a temperature, which is below about 20°

C.

20. A process of claim 1, wherein the sample, which remains in a liquid state, is heated to a temperature which is greater than about 20°

C. and less than about 80°

C.

21. A process of claim 1, wherein prior to step (c), the nucleic acid ions are extracted from the ion source by delayed extraction.

22. A process of claim 1, wherein the mass spectrometric format is selected from the group consisting of time-of-flight (TOF), quadrupole, magnetic sector, Fourier transform ion cyclotron resonance (FTICR), ion trap and combinations thereof.

23. A process of claim 22, wherein a mass spectrometry format is TOF and the TOF is linear.

24. A process of claim 22, wherein a mass spectrometry format is TOF and the TOF has a reflector.

25. A process of claim 22, wherein a mass spectrometry format is TOF and the TOF reflector has a linear field.

26. A process of claim 22, wherein a mass spectrometry format is TOF reflector has a nonlinear field.

27. A process of claim 22, wherein a mass spectrometry format is quadrupole and the quadrupole is single.

28. A process of claim 22, wherein a mass spectrometry format is quadrupole and the quadrupole is multiple.

29. A process of claim 22, wherein a mass spectrometry format is magnetic sector and the magnetic sector is single.

30. A process of claim 22, wherein a mass spectrometry format is magnetic sector and the magnetic sector is multiple.