Infrared matrix-assisted laser desorption/ionization mass spectrometric analysis of macromolecules

• US 6,558,902 B1
• Filed: 05/07/1999
• Issued: 05/06/2003
• Est. Priority Date: 05/07/1998
• 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
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/307,006
• Time in Patent Office: 1,460 Days
• Field of Search: 435/6, 435/91.2, 250/281, 436/94
• US Class Current: 506/6
• 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

Mixtures containing a biological macromolecule, such as a nucleic acid molecule or a polypeptide, and a liquid matrix, which absorbs infrared (IR) radiation, are provided. These mixtures are useful for analysis of the biological macromolecule by IR matrix assisted laser desorption/ionization (IR-MALDI) mass spectrometry. Also provided are processes for analyzing a biological macromolecule using IR-MALDI mass spectrometry. For example, processes for detecting the presence or identity of a biological macromolecule in a sample, or for sequencing a biological macromolecule are provided.

Claims

1. A method for performing matrix assisted laser desorption/ionization (MALDI) of a nucleic acid molecule 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, whereby the nucleic acid in the solution is desorbed and ionized.

2. The method of claim 1, further comprising, determining the mass of the nucleic acid by MALDI.

3. The method of claim 1, further comprising, determining the mass of the nucleic acid by MALDI and thereby detecting the presence of the nucleic acid in a sample.

4. A method of claim 1, wherein the liquid matrix has at least one of the following properties:

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

5. The method of claim 1, wherein the liquid matrix is sufficiently non-volatile to not evaporate during the illuminating, desorbing and ionizing step.

6. The method of claim 1, wherein the liquid matrix can form a glass when cooled and/or pressurized.

7. The method of claim 1, wherein the matrix comprises a sugar, a monosaccharide, or a polysaccharide.

8. The method of claim 1, wherein the matrix comprises a polyglycerol, sucrose, mannose, galactose, ethylene glycol, propylene glycol, trimethylolpropane, pentaerythritol, dextrose, methylglycoside or sorbitol, sucrose, mannose, triethanolamine, lactic acid, 3-nitrobenzylalcohol, diethanolamine, DMSO, nitrophenyloctylether (3-NPOE), 2,2′

dithiodiethanol, tetraethyleneglycol, dithiothreitol/erythritol (DTT/DTE), 2,3-dihydroxy-propyl-benzyl ether, α

-tocopherol, and thioglycerol.

9. A method of claim 1, wherein the liquid matrix contains at least one functional group that absorbs infrared radiation.

10. A method of claim 9, wherein the functional group is selected from the group consisting of nitro, sulfonyl, sulfonic acid, sulfonamide, nitrile, carbonyl, aldehyde, carboxylic acid, amide, ester, anhydride, ketone, amine, hydroxyl, an aromatic ring and a diene.

11. A method of claim 1, wherein the liquid matrix is selected from a group consisting of an alcohol, a carboxylic acid, a primary or secondary amide, a primary or secondary amine, a nitrile, hydrazine and hydrazide.

12. A method of claim 11, wherein the alcohol is selected from the group consisting of glycerol, 1,2- or 1,3-propane diol, 1,2-, 1,3- or 1,4-butane diol and triethanolamine.

13. A method of claim 11, wherein the carboxylic acid is selected from the group consisting of lactic acid, acetic acid, formic acid, propionic acid, butanoic acid, pentanoic acid, hexanoic acid and esters thereof.

14. A method of claim 11, wherein the amide is selected from the group consisting of acetamide, propanamide, butanamide, pentanamide and hexanamide, whether branched or unbranched.

15. A method of claim 11, wherein the amine is selected from the group consisting of propylamine, butylamine, pentylamine, hexylamine, heptylamine, diethylamine and dipropylamine.

16. A method of claim 4, wherein the liquid matrix is comprised of at least two liquids, each of which confers at least one of the properties.

17. A method of claim 1, wherein the liquid matrix comprises an additive.

18. A method of claim 17, 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.

19. A method of claim 17, wherein the additive increases the ionic strength of the matrix composition.

20. A method 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.

21. A method of claim 1, wherein the liquid matrix is treated by distillation or ion exchange.

22. A method of claim 1, wherein the liquid matrix is treated by further purification.

23. A method of claim 1, wherein the liquid matrix is selected from the group consisting of glycerol, lactic acid and triethanolamine.

24. A method of claim 23, wherein the liquid matrix is glycerol and the final analyte-to-glycerol molar ratio is about 10⁻

4 to about 10<sup>−

9</sup>.

25. A method 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).

26. A method of claim 1, wherein the nucleic acid is DNA.

27. A method of claim 26, wherein the DNA is less than or equal to about 2000 bases.

28. A method of claim 1, wherein the nucleic acid is RNA.

29. A method of claim 28, wherein the RNA is less than or equal to about 1200 bases.

30. A method of claim 1, wherein the nucleic acid comprises PNA.

31. A method of claim 1, wherein the nucleic acid comprises double-stranded nucleic acid.

32. A method of claim 1, wherein the nucleic acid comprises single-stranded nucleic acid.

33. A method 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.

34. A method of claim 1, wherein the infrared radiation is generated in pulses having a duration in the range of about 500 ps to about 500 ns.

35. A method of claim 34, wherein the pulse duration is less than 200 ns.

36. A method of claim 35, wherein the pulse duration is less than 100 ns.

37. A method of claim 1, wherein the infrared radiation is generated from a source selected from the group consisting of 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.

38. A method of claim 1, wherein the solution contains less than about 10 pmoles of nucleic acid.

39. A method of claim 1, wherein all or a portion of the method is automated.

40. A method of claim 1, wherein the solution is cooled to a temperature, which is below about 20°

C.

41. A method of claim 1, wherein the solution is heated to a temperature which is greater than about 20°

C. and less than about 80°

C.

42. A method of claim 1, wherein the solution is cooled, whereby the matrix forms a glass.

43. A method of claim 42, wherein the glass is glassy water.

44. A method of claim 1, wherein the matrix comprises glycerol and the glycerol and nucleic acid solution is cooled, whereby the glycerol freezes.

45. A method of claim 1, wherein ions from the desorbed and ionized nucleic acid are extracted from an ion source by delayed extraction.

92. A method for determining the size of a primer extension product, comprising:

(a) hybridizing a primer with a target nucleic acid, where the primer (i) is complementary to the target nucleic acid;

(ii) has a first region containing the 5′

end of the primer and an immobilization attachment site, and (iii) has a second region containing the 3′

end of the primer, where the 3′

end is capable of serving as a priming site for enzymatic extension and where the second region contains a selected cleavable site;

(b) extending the primer enzymatically to generate a polynucleotide mixture containing an extension product composed of the primer and an extension segment;

(c) cleaving the extension product at the cleavable site to release the extension segment, where prior to the cleaving the primer is immobilized at the immobilization attachment site; and

(d) sizing the extension segment by the method of claim 1, whereby the cleaving is effective to increase the read length of the extension segment relative to the read length of the product of (b).

93. A method for determining the size of a primer extension product, comprising:

(a) combining first and second primers with a target nucleic acid under conditions that promote the hybridization of the primers to the nucleic acid, thus generating primer/nucleic acid complexes, where the first primer (i) is complementary to the target nucleic acid;

(ii) has a first region containing the 5′

end of the primer and an immobilization attachment site, and (iii) has a second region containing the 3′

end of the primer, where the 3′

end is capable of serving as a priming site for enzymatic extension and where the second region contains a cleavable site, and where the second primer is homologous to the target nucleic acid;

(b) converting the primer/nucleic acid complexes to double-stranded fragments in the presence of a suitable polymerase and all four dNTPs;

(c) amplifying the primer-containing fragments by successively repeating the steps of (i) denaturing the double-stranded fragments to produce single-strand fragments, (ii) hybridizing the single strands with the primers to form strand/primer complexes, (iii) generating double-stranded fragments from the strand/primer complexes in the presence of DNA polymerase and all four dNTPs, and (iv) repeating steps (i) to (iii) until a desired degree of amplification has been achieved;

(d) denaturing the amplified fragments to generate a mixture including a product composed of the first primer and an extension segment;

(e) immobilizing amplified fragments containing the first primer, utilizing the immobilization attachment site, and removing non-immobilized amplified fragments;

(f) cleaving the immobilized fragments at the cleavable site to release the extension segment; and

(g) sizing the extension segment by the method of claim 1, whereby the cleaving is effective to increase the read length of the extension segment relative to the read length of the product of (d).

94. A method for determining the DNA sequence of a target DNA sequence, comprising:

(a) hybridizing a primer with a target DNA, where the primer (i) is complementary to the target DNA;

(ii) has a first region containing the 5′

end of the primer and an immobilization attachment site, and (iii) has a second region containing the 3′

end of the primer, where the 3′

end is capable of serving as a priming site for enzymatic extension and where the second region contains a cleavable site;

(b) extending the primer with an enzyme in the presence of a first of four different dideoxy nucleotides to generate a mixture of primer extension products each product containing a primer and an extension segment;

(c) cleaving at the cleavable site to release the extension segments, where prior to the cleaving the primers are immobilized at the immobilization attachment sites;

(d) sizing the extension segments by the method of claim 1, whereby the cleaving is effective to increase the read length of the extension segment relative to the read length of the product of (b);

(e) repeating steps (a) through (d) with a second, third, and fourth of the four different dideoxynucleotides; and

(f) determining the DNA sequence of the target DNA by comparison of the sizes of the extension segments obtained from each of the four extension reactions.

95. A method for determining the DNA sequence of a target DNA sequence, comprising:

(a) hybridizing a primer with a target DNA, where the primer (i) is complementary to the target DNA;

(ii) has a first region containing the 5′

end of the primer and an immobilization attachment site, and (iii) has a second region containing the 3′

end of the primer, where the 3′

end is capable of serving as a priming site for enzymatic extension and where the second region contains a cleavable site;

(b) extending the primer with an enzyme in the presence of a first of four different deoxynucleoside α

-thiotriphosphate analogs (dNTPα

S) to generate a mixture of primer extension products containing phosphorothioate linkages;

(c) treating the primer extension products with a reagent that cleaves specifically at the phosphorothioate linkages, where the treating is carried out under conditions producing limited cleavage, resulting in the production of a group of primer extension degradation products;

(d) washing the primer extension degradation products, where prior to the washing, the primer extension degradation products are immobilized at the immobilization attachment sites, each immobilized primer extension degradation product containing a primer and an extension segment, where the washing is effective to remove non-immobilized species;

(e) cleaving at the cleavable site to release the extension segments;

(f) sizing the extension segments by the method of claim 1, whereby the cleaving is effective to increase the read length of any given extension segment relative to the read length of its corresponding primer extension degradation product;

(g) repeating steps (a) through (f) with a second, third, and fourth of the four different dNTPα

Ss; and

(h) determining the DNA sequence of the target DNA by comparison of the sizes of the extension segments obtained from each of the four extension reactions.

96. A method for determining the size of a primer extension product, comprising:

(a) hybridizing a primer with a target nucleic acid, where the primer (i) is complementary to the target nucleic acid;

(ii) has a first region containing the 5′

end of the primer, and (iii) has a second region containing the 3′

end of the primer, where the 3′

end is capable of serving as a priming site for enzymatic extension and where the second region contains a selected cleavable site;

(b) extending the primer enzymatically to generate a polynucleotide mixture containing an extension product composed of the primer and an extension segment;

(c) cleaving the extension product at the cleavable site to release the extension segment; and

(d) sizing the extension segment by the method of claim 1, whereby the cleaving is effective to increase the read length of the extension segment relative to the read length of the product of (b).

97. A method for determining the size of a primer extension product, comprising:

(a) hybridizing a primer with a target nucleic acid, where the primer (i) is complementary to the target nucleic acid;

(ii) has a first region containing the 5′

end of the primer, and an immobilization attachment site, where the immobilization attachment site of the primer is composed of a series of bases complementary to an intermediary oligonucleotide, and (iii) has a second region containing the 3′

end of the primer, where the 3′

end is capable of serving as a priming site for enzymatic extension and where the second region contains a selected cleavable site;

(b) extending the primer enzymatically to generate a polynucleotide mixture containing an extension product composed of the primer and an extension segment;

(c) cleaving the extension product at the cleavable site to release the extension segment, where prior to the cleaving the primer is immobilized by specific hybridization of the immobilization attachment site to the intermediary oligonucleotide bound to a solid support; and

(d) sizing the extension segment by the method of claim 1, whereby the cleaving is effective to increase the read length of the extension segment relative to the read length of the product of (b).

98. A method for determining the size of a primer extension product, comprising:

(a) combining first and second primers with a target nucleic acid, under conditions that promote hybridization of the primers to the nucleic acid, generating primer/nucleic acid complexes, where the first primer (i) has a 5′

end and a 3′

end, (ii) is complementary to the target nucleic acid, (iii) has a first region containing the 5′

end of the first primer and (iv) has a second region containing the 3′

end of the first primer, where the 3′

end is capable of serving as a priming site for enzymatic extension and where the second region contains a cleavable site, and where the second primer (i) has a 5′

end and a 3′

end, (ii) is homologous to the target nucleic acid, (iii) has a first segment containing the 3′

end of the second primer, and (iv) has a second segment containing the 5′

end of the second primer and an immobilization attachment site;

(b) converting the primer/nucleic acid complexes to double-stranded fragments in the presence of a DNA polymerase and deoxynucleoside triphosphates;

(c) amplifying the primer-containing fragments by successively repeating the steps of (i) denaturing the double-stranded fragments to produce single-stranded fragments, (ii) hybridizing the single stranded fragments with the first and second primers to form strand/primer complexes, (iii) generating amplification products from the strand/primer complexes in the presence of DNA polymerase an deoxynucleoside triphosphates, and (iv) repeating steps (i) to (iii) until a desired degree of amplification has been achieved;

(d) immobilizing amplification products containing the second primer via the immobilization attachment site;

(e) removing non-immobilized amplified fragments;

(f) cleaving the immobilized amplification products at the cleavable site, to generate a mixture including a double-stranded product;

(g) denaturing the double-stranded product to release the extension segment; and

(h) sizing the extension segment by the method of claim 1, whereby the cleaving is effective to increase the read length of the extension segment relative to the read length of the amplified strand-primer complexes of (c).

99. A method for determining a single base fingerprint of a target DNA sequence, comprising:

(a) hybridizing a primer with a target DNA, where the primer (i) is complementary to the target DNA;

(ii) has a first region containing the 5′

end of the primer and an immobilization attachment site, and (iii) has a second region containing the 3′

end of the primer, where the 3′

end is capable of serving as a priming site for enzymatic extension and where the second region contains a selected cleavable site;

(b) extending the primer with an enzyme in the presence of a dideoxynucleoside triphosphate corresponding to the single base, to generate a polynucleotide mixture of primer extension products, each product containing a primer and an extension segment;

(c) cleaving the extension products at the cleavable site to release the extension segments, where prior to the cleaving the primers are immobilized at the immobilization attachment sites;

(d) sizing the extension segments by the method of claim 1, whereby the cleaving is effective to increase the read length of any given extension segment relative to the read length of its corresponding primer extension product of (b); and

(e) determining the positions of the single base in the target DNA by comparison of the sizes of the extension segments.

100. A method for an adenine fingerprint of a target DNA sequence, comprising:

(a) hybridizing a primer with a DNA target, where the primer (i) is complementary to the target DNA;

(ii) has a first region containing the 5′

end of the primer and an immobilization attachment site, and (iii) has a second region containing the 3′

end of the primer, where the 3′

end is capable of serving as a priming site for enzymatic extension and where the second region contains a selected cleavable site;

(b) extending the primer with an enzyme in the presence of deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxyuridine triphosphate (dUTP), to generate a polynucleotide mixture of primer extension products containing dUTP at positions corresponding to dATP in the target, each product containing a primer and an extension segment;

(c) treating the primer extension products with uracil DNA-glycosylase to fragment specifically at dUTP positions to produce a set of primer extension degradation products;

(d) washing the primer extension degradation products, where prior to the washing, the primer extension degradation products are immobilized at the immobilization attachment sites, each immobilized primer extension degradation product containing a primer and an extension segment, where the washing is effective to remove non-immobilized species;

(e) cleaving the immobilized primer extension degradation products at the cleavable site to release the extension segments;

(f) sizing the extension segments by the method of claim 1, whereby the cleaving is effective to increase the read length of any given extension segment relative to the read length of its corresponding primer extension degradation product; and

(g) determining the positions of adenine in the target DNA by comparison of the sizes of the released extension segments.

101. A method of detecting mutations in a target nucleic acid, comprising:

a) obtaining from the target nucleic acid a set of nonrandom length fragments (NLFs) in single-stranded form, wherein the set comprises NLFs derived from one of either the positive or the negative strand of the target nucleic acid or the set is a subset of single-stranded NLFs derived from the positive and the negative strand of the target nucleic acid; and

b) determining masses of the members of the set by the method of claim 1.

102. A method of detecting mutations in a target nucleic acid, comprising:

a) nonrandomly fragmenting the target nucleic acid with one or more restriction endonucleases to form a set of double-stranded NLFs, wherein the nonrandomly fragmenting further comprises using volatile salts in a restriction buffer; and

b) determining masses of the members of the set of double-stranded NLFs by the method of claim 1.

103. A method of detecting mutations in a double-stranded target nucleic acid comprising:

a) nonrandomly fragmenting the target nucleic acid using one or more restriction endonucleases to form a first set of nonrandom length fragments (NLFs);

b) hybridizing members of the first set of NLFs to a set of wild type probes;

c) nonrandomly fragmenting one or more members of the set of NLFs with one or more mutation-specific cleaving reagents that specifically cleave at any regions of nucleotide mismatch that form between members of the first set of NLFs and complementary members of the set of wild type probes, wherein the nonrandomly fragmenting step forms a second set of NLFs; and

d) determining masses of members of the second set of NLFs by the method of claim 1.

104. A method of detecting mutations in a target nucleic acid, comprising:

a) nonrandomly fragmenting the target nucleic acid, using a mixture comprising one or more volatile salts to form a set of nonrandom length fragments (NLFs); and

b) determining masses of members of the set of NLFs by the method of claim 1.

105. The method of claim 1, further comprising washing the nucleic acid sample with a mixture of volatile salts, and evaporating the mixture of volatile salts from the sample, thereby decreasing background noise.

106. A method for identifying DNA tandem nucleotide repeat alleles at a DNA tandem nucleotide repeat locus in a target nucleic acid by mass spectrometry, the method comprising:

a) obtaining a target nucleic acid comprising a DNA tandem nucleotide repeat region;

b) extending the target nucleic acid using one or more primers to obtain a limited size range of nucleic acid extension products, wherein one or more primers are complementary to a sequence flanking the DNA tandem nucleotide repeat of the locus; and

c) determining the mass of the nucleic acid extension products by the method of claim 1.

107. A method for multiplexing the identification of more than one DNA tandem nucleotide repeat regions from more than one DNA tandem nucleotide repeat loci by mass spectrometry, which method comprises:

a) obtaining more than one nucleic acid extension products by extending one or more primers complementary to sequences flanking the DNA tandem nucleotide repeat regions; and

b) determining the mass of the more than one nucleic acid extension products simultaneously by the method of claim 1, wherein the nucleic acid extension products have overlapping allelic mass ranges.

108. A method for multiplexing the identification of more than one DNA tandem nucleotide repeat regions for more than one DNA tandem nucleotide repeat loci, which method comprises:

a) obtaining more than one nucleic acid amplification products by amplifying two or more primers complementary to sequences flanking the DNA tandem nucleotide repeat regions; and

b) determining the masses of more than one nucleic acid amplification products simultaneously by the method of claim 1, wherein the nucleic acid extension products have overlapping allelic mass ranges.

109. A method for identifying DNA tandem nucleotide repeat alleles at a DNA tandem nucleotide repeat locus in a target nucleic acid by mass spectrometry, the method comprising

a) obtaining a target nucleic acid comprising a DNA tandem nucleotide repeat region;

b) extending the target nucleic acid using one or more primers to obtain a limited size range of nucleic acid extension products, wherein one or more primers are complementary to a sequence flanking the DNA tandem nucleotide repeat of the locus; and

c) determining the mass of the nucleic acid extension products by the method of claim 1.

110. The method of claim 109, wherein a 3′

end of one or more primers immediately flanks a DNA tandem nucleotide repeat region.

111. The method of claim 109, wherein one or more primers comprise a sequence complementary to up to one tandem repeat of the DNA tandem nucleotide repeat locus.

112. The method of claim 111, wherein one or more primers comprise a sequence complementary to up to two tandem repeats of the DNA tandem nucleotide repeat locus.

113. The method of claim 112, wherein one or more primers comprise a sequence complementary to up to three tandem repeats of the DNA tandem nucleotide repeat locus.

114. The method of claim 109, wherein at least one primer comprises a cleavable site.

115. The method of claim 114, wherein the cleavable site comprises a recognition site for a restriction endonuclease, an exonuclease blocking site, or a chemically cleavable site.

116. The method of claim 114, wherein at least one primer is capable of attaching to a solid support.

117. The method of claim 116, wherein at least one primer comprises biotin or digoxigenin.

118. The method of claim 109, wherein the extension of at least one primer is terminated using a chain termination reagent.

119. The method of claim 118, wherein the chain termination reagent is a dideoxynucleotide triphospate.

46. A method for analyzing a nucleic acid in a sample by mass spectrometry, comprising the steps of:

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

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

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

47. The method of claim 46, wherein the liquid matrix is sufficiently non-volatile to not evaporate during the illuminating, desorbing and ionizing step.

48. The method of claim 46, wherein the liquid matrix can form a glass when cooled and/or pressurized.

49. The method of claim 46, wherein the matrix comprises a sugar, a monosaccharide, or a polysaccharide.

50. The method of claim 46, wherein the matrix comprises a polyglycerol, sucrose, mannose, galactose, ethylene glycol, propylene glycol, trimethylolpropane, pentaerythritol, dextrose, methylglycoside or sorbitol, sucrose, mannose, triethanolamine, lactic acid, 3-nitrobenzylalcohol, diethanolamine, DMSO, nitrophenyloctylether (3-NPOE), 2,2′

dithiodiethanol, tetraethyleneglycol, dithiothreitol/erythritol(DTT/DTE), 2,3-dihydroxy-propyl-benzyl ether, α

-tocopherol, and thioglycerol.

51. A method of claim 46, wherein the liquid matrix has at least one of the following 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.

52. A method of claim 46, wherein the liquid matrix contains at least one functional group that absorbs infrared radiation.

53. A method of claim 52, wherein the functional group is selected from the group consisting of nitro, sulfonyl, sulfonic acid, sulfonamide, nitrile, carbonyl, aldehyde, carboxylic acid, amide, ester, anhydride, ketone, amine, hydroxyl, an aromatic ring and a diene.

54. A method of claim 46, wherein the liquid matrix is selected from a group consisting of an alcohol, a carboxylic acid, a primary or secondary amide, a primary or secondary amine, a nitrile, hydrazine and hydrazide.

55. A method of claim 54, wherein the alcohol is selected from the group consisting of glycerol, 1,2- or 1,3-propane diol, 1,2-, 1,3- or 1,4-butane diol and triethanolamine.

56. A method of claim 54, wherein the carboxylic acid is selected from the group consisting of lactic acid, acetic acid, formic acid, propionic acid, butanoic acid, pentanoic acid, hexanoic acid and esters thereof.

57. A method of claim 54, wherein the amide is selected from the group consisting of acetamide, propanamide, butanamide, pentanamide and hexanamide, whether branched or unbranched.

58. A method of claim 54, wherein the amine is selected from the group consisting of propylamine, butylamine, pentylamine, hexylamine, heptylamine, diethylamine and dipropylamine.

59. A method of claim 51, wherein the liquid matrix is comprised of at least two liquids, each of which confers at least one of the properties.

60. A method of claim 46, wherein the liquid matrix comprises an additive.

61. A method of claim 60, 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.

62. A method of claim 60, wherein the additive increases the ionic strength of the matrix composition.

63. A method of claim 46, 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.

64. A method of claim 46, wherein the liquid matrix is treated by distillation or ion exchange.

65. A method of claim 46, wherein the liquid matrix is treated by further purification.

66. A method of claim 46, wherein the liquid matrix is selected from the group consisting of glycerol, lactic acid and triethanolamine.

67. A method of claim 46, wherein the liquid matrix is glycerol and the final analyte-to-glycerol molar ratio is about 10⁻

4 to about 10<sup>−

9</sup>.

68. A method of claim 46, 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).

69. A method of claim 46, wherein the nucleic acid is DNA.

70. A method of claim 69, wherein the DNA is less than or equal to about 2000 bases.

71. A method of claim 46, wherein the nucleic acid is RNA.

72. A method of claim 71, wherein the RNA is less than or equal to about 1200 bases.

73. A method of claim 46, wherein the nucleic acid comprises PNA.

74. A method of claim 46, wherein the nucleic acid comprises double-stranded nucleic acid.

75. A method of claim 46, wherein the nucleic acid comprises single-stranded nucleic acid.

76. A method of claim 46, wherein the infrared radiation is of a wavelength in the range of from about 2.5 μ

m to about 12 μ

m.

77. A method of claim 46, wherein the infrared radiation is generated in pulses having a width in the range of about 500 ps to about 500 ns.

78. A method of claim 46, wherein the infrared radiation is generated from a source selected from the group consisting of 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.

79. A method of claim 46, wherein the sample contains less than about 10 pmoles of nucleic acid.

80. A method of claim 46, wherein all or a portion of the method is automated.

81. A method of claim 46, wherein the sample is cooled to a temperature that is below about 20°

C.

82. A method of claim 46, wherein the sample is heated to a temperature which is greater than about 20°

C. and less than about 80°

C.

83. A method of claim 46, wherein the matrix and nucleic acid mixture are cooled, whereby the matrix forms a glass.

84. A method of claim 83, wherein the glass is glassy water.

85. A method of claim 46, wherein the matrix comprises glycerol and the glycerol and sample mixture are cooled, whereby the glycerol freezes.

86. A method of claim 46, wherein prior to step (c), ions from the ionized nucleic acid are extracted from an ion source by delayed extraction.

87. A method of claim 46, 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), and ion trap or a combination thereof.

88. A method of claim 87, wherein the TOF is linear, or the TOF has a reflector.

89. A method of claim 88, wherein the TOF reflector has a linear field or a nonlinear field.

90. A method of claim 87, wherein the quadrupole is single or the quadrupole is multiple.

91. A method of claim 87, wherein the magnetic sector is single or the magnetic sector is multiple.

120. A method for detecting a target molecule, comprising:

(a) obtaining a target molecule;

(b) amplifying the target molecule to produce an amplified target molecule;

(c) obtaining a probe comprising a reactive group, a release group and a mass label;

(d) hybridizing the amplified target molecule to the probe to produce a probe;

amplified target molecule complex;

(e) releasing the mass label from the probe;

amplified target molecule complex to obtain a released mass label;

(f) depositing a solution containing the released mass label and a liquid matrix on a substrate, thereby forming a homogeneous, thin layer of a mass label/liquid matrix solution;

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

(h) determining the mass of the released mass label by matrix assisted laser desorption/ionization (MALDI) mass spectrometry.

121. The method of claim 120, wherein prior to step (f), the mass label is mixed with the liquid matrix.

122. The method of claim 121, wherein the liquid matrix is glycerol.

123. A method for detecting a target molecule, comprising:

(a) obtaining a probe comprising a reactive group, a release group and a mass label;

(b) obtaining a target molecule;

(c) contacting the target molecule with the probe to produce a probe;

target molecule complex;

(d) releasing the mass label from the probe;

target molecule complex;

(e) depositing a solution containing the released mass label and a liquid matrix on a substrate, thereby forming a homogeneous, thin layer of a mass label/liquid matrix solution;

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

(g) determining the mass of the released mass label by matrix assisted laser desorption/ionization (MALDI) mass spectrometry.

124. The method of claim 123, wherein the mass label is nonvolatile and the mass label is selectively released from the probe:

target molecule complex.

125. The method of claim 123, wherein prior to step (e), the mass label is mixed with a liquid matrix.

126. The method of claim 124, wherein the matrix is glycerol.

127. A method for multiplexing the detection of a target molecule, comprising:

(a) obtaining a plurality of probes, each comprising a reactive group, a release group and a mass label;

(b) contacting the target molecule with the plurality of probes to produce probe;

target molecule complexes, wherein the target molecule is attached to the reactive group of the probe;

(c) releasing the mass labels from the probe;

target molecule complexes to produce released mass labels;

(d) depositing a solution containing the released mass labels and a liquid matrix on a substrate, thereby forming a homogeneous, thin layer of a mass label/liquid matrix solution;

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

(f) determining the masses of the released mass labels by matrix assisted laser desorption/ionization (MALDI) mass spectrometry, wherein each reactive group in a probe;

target molecule complex is associated with a unique set of mass labels.

128. The method of claim 127, wherein prior to step (d), the mass labels are mixed with a liquid matrix.

129. The method of claim 127, wherein the matrix is glycerol.

130. A method for multiplexing the detection of a plurality of target molecules, comprising:

(a) obtaining a plurality of probes, each comprising a reactive group, a release group and a mass label;

(b) contacting the plurality of target molecules with the plurality of probes to produce probe;

target molecule complexes, wherein target molecules are attached to the reactive groups of the probes;

(c) releasing the mass labels from the probe;

target molecule complexes to produce released mass labels;

(d) depositing a solution containing the released mass labels and a liquid matrix on a substrate, thereby forming a homogeneous, thin layer of a mass label/liquid matrix solution;

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

(f) determining the masses of the released mass labels by matrix assisted laser desorption/ionization (MALDI) mass spectrometry, wherein each reactive group specific for a particular target molecule is associated with a unique mass label.

131. The method of claim 130, wherein prior to step (d), the mass labels are mixed with a liquid matrix.

132. The method of claim 131, wherein the matrix is glycerol.

133. A method for detecting a target biological macromolecule, comprising the steps of:

a) preparing a mixture, comprising a biological macromolecule and a liquid matrix, which absorbs infrared radiation; and

b) performing IR-MALDI mass spectrometry on the mixture to identify the target biological macromolecule in the mixture, thereby detecting the target biological macromolecule.

134. The method of claim 133, wherein the target biological macromolecule is in a biological sample, whereby detection of the target biological macromolecule identifies the presence of the target biological macromolecule in the biological sample.

135. The method of claim 133, wherein the biological macromolecule is a nucleic acid.

136. The method of claim 133, wherein the biological macromolecule is a polypeptide.

137. The method of claim 133, wherein the biological macromolecule is selected from the group consisting of a carbohydrate, lipid, a nucleoprotein, a proteoglycan, and a macromolecular complex.

138. The method of claim 133, wherein the target biological macromolecule is immobilized on a solid support.

139. The method of claim 138, wherein the target biological macromolecule is immobilized to the solid support via a reversible linkage.

140. The method of claim 138, wherein the target biological macromolecule is immobilized to the solid support via a photocleavable bond.

141. The method of claim 139, wherein the reversible linkage is a thiol linkage or an ionic bond.

142. The method of claim 138, wherein the target biological macromolecule is cleaved from the support during the step of performing IR-MALDI mass spectrometry.

143. The method of claim 138, wherein the solid support is selected from the group consisting of a bead, a flat surface, a chip, a capillary, a pin, a comb, a wafer, a wafer with an arrow of nano-wells or pits, the terminus of a fiber optic cable, a support with a surface that comprises hydrophobic regions and a support with a surface that comprises hydrophilic regions, whereby the target molecule is constrained to a locus on the support.

144. The method of claim 138, wherein the solid support is a material selected from the group consisting of a metal, a ceramic, a plastic, a resin, a gel, and a membrane.

145. The method of claim 138, wherein the support is a silicon wafer, and wherein the target biological macromolecule is immobilized in an array.

146. The method of claim 135, wherein a target nucleic acid is immobilized by hybridization to a complementary capture nucleic acid molecule, which is immobilized to a solid support.

147. The method of claim 133, wherein the target biological macromolecule is conditioned prior to the step of performing IR-MALDI mass spectrometry.

148. The method of claim 147, wherein the target biological macromolecule is conditioned by ion exchange.

149. The method of claim 135, wherein the target nucleic acid is conditioned by a method selected from the group consisting of phosphodiester backbone modification effected by cation exchange;

contact with an alkylating agent or trialkylsilyl chloride;

incorporation of at least one nucleotide that reduces sensitivity for depurination in the target nucleic acid;

incorporation of at least one mass modified nucleotide in the target nucleic acid; and

hybridization of a tag probe to a portion of a nucleic acid molecule that contains the target nucleic acid but is distinct from a target nucleic acid sequence.

150. The method of claim 136, wherein the target polypeptide is obtained by in vitro translation, or by in vitro transcription followed by translation, of a nucleic acid encoding the target polypeptide.

151. The method of claim 150, wherein the nucleic acid encoding the target polypeptide further comprises a nucleotide sequence encoding a second polypeptide.

152. The method of claim 136, wherein the target polypeptide comprises a tag.

153. A method for detecting the presence of a target nucleic acid sequence in a biological sample containing nucleic acid molecules, comprising the steps of:

a) contacting the nucleic acid molecules with a detector oligonucleotide, which can hybridize to a target nucleic acid sequence present in the biological sample;

b) preparing a mixture for IR-MALDI, comprising the product of step a) and a liquid matrix, which absorbs infrared radiation;

c) identifying duplex nucleic acid molecules in the mixture by IR-MALDI mass spectrometry, thereby detecting presence of the target nucleic acid sequence in the biological sample.

154. The method of claim 153, further comprising, prior to step a), a step of amplifying the nucleic acid molecules in the biological sample.

155. A method for detecting the presence of a target nucleic acid sequence in a biological sample containing nucleic acid molecules, comprising the steps of:

a) specifically digesting the nucleic acid molecules using at least one appropriate nuclease, thereby producing digested fragments;

b) hybridizing the digested fragments with complementary capture nucleic acid sequences, which are immobilized on a solid support and can hybridize to a digested fragment of a target nucleic acid to produce immobilized fragments;

c) preparing a mixture for IR-MALDI, comprising the immobilized fragments and a liquid matrix, which absorbs infrared radiation; and

d) identifying immobilized fragments by IR-MALDI mass spectrometry, thereby detecting the presence of the target nucleic acid sequence in the biological sample.

156. The method of claim 155, further comprising, prior to step a), a step of amplifying the nucleic acid molecules in the biological sample.

157. A method for detecting a target nucleic acid sequence, comprising the steps of:

a) performing at least one hybridization on a nucleic acid molecule containing the target nucleic acid sequence with a set of ligation educts and a thermostable DNA ligase;

b) preparing a mixture for IR-MALDI, comprising the product of step a) and a liquid matrix, which absorbs infrared radiation; and

c) identifying a ligation product in the mixture by IR-MALDI mass spectrometry, thereby detecting the target nucleic acid sequence.

158. A method for detecting the presence of a target nucleic acid in a biological sample containing nucleic acid molecules, comprising the steps of:

a) performing on the nucleic acid molecules, a first polymerase chain reaction using a first set of primers, which are capable of amplifying a portion of a nucleic acid molecule containing the target nucleic acid, thereby producing a first amplification product;

b) preparing a mixture for IR-MALDI, comprising the first amplification product and a liquid matrix, which absorbs infrared radiation; and

c) detecting the first amplification product in the mixture by IR-MALDI mass spectrometry, thereby detecting the presence of the target nucleic acid in the biological sample.

159. The method of claim 158, wherein prior to step b), a second polymerase chain reaction is performed on the first amplification product using a second set of primers, which are capable of amplifying at least a portion of the first amplification product, which contains the target nucleic acid.

160. A method for determining the identity of a target nucleotide, comprising the steps of:

a) hybridizing a nucleic acid molecule containing the target nucleotide with a primer oligonucleotide that is complementary to the nucleic acid molecule at a site adjacent to the target nucleotide, thereby producing a hybridized nucleic acid molecule;

b) contacting the hybridized nucleic acid molecule with a complete set of dideoxynucleosides or 3′

-deoxynucleoside triphosphates and a DNA dependent DNA polymerase, so that only the dideoxynucleosides or 3′

-deoxynucleoside triphosphate that is complementary to the target nucleotide is extended onto the primer, thereby producing an extended primer;

c) preparing a mixture for IR-MALDI, comprising the extended primer and a liquid matrix, which absorbs infrared radiation; and

d) detecting the extended primer in the mixture by IR-MALDI mass spectrometry, thereby determining the identity of the target nucleotide.

161. A method for detecting the presence or absence of a mutation in a target nucleic acid sequence, comprising the steps of:

a) hybridizing a nucleic acid molecule containing the target nucleic acid sequence with at least one primer, the primer having 3′

terminal base complementarity to the target nucleic acid sequence, thereby producing a hybridized product;

b) contacting the hybridized product with an appropriate polymerase enzyme and sequentially with one of the four nucleoside triphosphates;

c) preparing a mixture for IR-MALDI, comprising the product of step b) and a liquid matrix, which absorbs infrared radiation; and

d) detecting the product of step b) in the mixture by IR-MALDI mass spectrometry, wherein the molecular weight of the product indicates the presence or absence of a mutation next to the 3′

end of the primer in the target nucleic acid molecule.

162. The method of claim 161, wherein the nucleic acid molecule containing the target nucleic acid sequence is immobilized to a solid support.

163. The method of claim 161, wherein, prior to step a), the nucleic acid molecule containing the target nucleic acid sequence is amplified.

164. A method for detecting a mutation in a target nucleic acid, comprising the steps of:

a) hybridizing the target nucleic acid with an oligonucleotide probe, to produce a hybridized target nucleic acid, wherein a mismatch is formed at the site of a mutation;

b) contacting the hybridized target nucleic acid with a single strand specific endonuclease;

c) preparing a mixture for IR-MALDI, comprising the product of step b) and a liquid matrix, which absorbs infrared radiation; and

d) analyzing the mixture by IR-MALDI mass spectrometry, wherein the presence of more than one fragment of the target nucleic acid in the mixture detects a mutation the target nucleic acid.

165. A method for identifying the absence or presence of a mutation in a target nucleic acid sequence, comprising the steps of:

a) performing at least one hybridization on a nucleic acid molecule containing the target nucleic acid sequence with a set of ligation educts and a DNA ligase;

b) preparing a mixture for IR-MALDI, comprising the product of step a) and a liquid matrix, which absorbs infrared radiation; and

c) analyzing the mixture by IR-MALDI mass spectrometry,

wherein detecting a ligation product in the mixture identifies the absence of a mutation in the target nucleic acid sequence, and

wherein detecting only the set of ligation educts in the mixture identifies the presence of a mutation in the target nucleic sequence.

166. A method for determining the identity of each target biological macromolecule in a plurality of target biological macromolecules, comprising the steps of:

a) preparing a mixture for IR-MALDI, comprising a plurality of differentially mass modified target biological macromolecules and a liquid matrix, which absorbs infrared radiation;

b) determining the molecular mass of each differentially mass modified target biological macromolecule in the plurality by IR-MALDI mass spectrometry; and

c) comparing the molecular mass of each differentially mass modified target biological macromolecule in the plurality with the molecular mass of a corresponding known biological macromolecule, thereby determining the identity of each target biological macromolecule in the plurality of target biological macromolecules or fragments thereof.

167. The method of claim 166, wherein each target biological macromolecule in the plurality is a fragment of a biological macromolecule, each fragment prepared by contacting the biological macromolecule with at least one agent that cleaves a bond involved in the formation of the biological macromolecule.

168. A method for determining the sequence of a target biological macromolecule, comprising the steps of:

a) generating at least two biological macromolecule fragments from the target biological macromolecule;

b) preparing a mixture for IR-MALDI, comprising the biological macromolecule fragments and a liquid matrix, which absorbs infrared radiation; and

c) analyzing the biological macromolecule fragments in the mixture by IR-MALDI mass spectrometry, thereby determining the sequence of the target nucleic acid molecule.

169. A method of determining the subunit sequence of at least one species of target biological macromolecule, i, comprising the steps of:

a) contacting the species of target biological macromolecule with at least one agent that cleaves a bond involved in the formation of the target biological macromolecule such that each bond in involved in the formation of the target biological macromolecule is cleaved, thereby producing a nested set of deletion fragments of the species of biological macromolecule;

b) preparing a mixture for IR-MALDI, comprising the nested set of deletion fragments and a liquid matrix, which absorbs infrared radiation; and

c) determining the molecular mass of each deletion fragment in mixture by IR-MALDI mass spectrometry, thereby determining the subunit sequence of the species of target biological macromolecule.

170. The method of claim 169, wherein the agent that cleaves is an agent that cleaves the target biological macromolecule unilaterally from a terminus.

171. The method of claim 170, wherein the target biological macromolecule is a nucleic acid and the agent that cleaves is an exonuclease.

172. The method of claim 170, wherein the target biological macromolecule is a polypeptide and the agent that cleaves is an exopeptidase.

173. The method of claim 170, wherein at least one species of target biological macromolecule comprises i+1 species of target biological macromolecules, and wherein each species of target biological macromolecule is differentially mass modified such that a deletion fragment of each species of target biological macromolecule can be distinguished from a deletion fragment of every other target biological macromolecule by IR-MALDI mass spectrometry.

176. The method of claim 173, wherein i+1 species of nucleic acids are concurrently sequenced by multiplex mass spectrometric nucleic acid sequencing employing i+1 primers, wherein one of the i+1 primers is an unmodified primer or a mass modified primer and the other i primers are mass modified primers, and each of the i+1 primers can be distinguished from the other by IR-MALDI mass spectroetry.

174. A method of determining the nucleotide sequence of at least one species of nucleic acid, i, comprising the steps of:

a) synthesizing complementary nucleic acids, which are complementary to the species of nucleic acid to be sequenced, starting from an oligonucleotide primer and in the presence of chain terminating nucleoside triphosphates, thereby producing four sets of base-specifically terminated complementary polynucleotide fragments;

b) preparing a mixture for IR-MALDI, comprising the four sets of polynucleotide fragments and a liquid matrix, which absorbs infrared radiation; and

c) determining the molecular weight value of each polynucleotide fragment by IR-MALDI mass spectrometry; and

d) determining the nucleotide sequence of the species of nucleic acid by aligning the molecular weight values-according to molecular weight.

175. The method of claim 174, wherein the species of nucleic acid is RNA, the chain terminating nucleoside triphosphates are ribonucleotide triphosphates or derivatives thereof, and the oligonucleotide primer is an initiator oligonucleotide.

177. A method for determining a sequence of a target nucleic acid, comprising the steps of:

a) hybridizing at least one partially single stranded target nucleic acid to one or more nucleic acid probes, each probe comprising a double stranded portion, a single stranded portion, and a determinable variable sequence within the single stranded portion, thereby producing at least one hybridized target nucleic acid;

b) preparing a mixture for IR-MALDI, comprising the hybridized target nucleic acid and a liquid matrix, which absorbs infrared radiation;

c) determining a sequence of the hybridized target nucleic acid by IR-MALDI mass spectrometry based on the determinable variable sequence of the probe to which the target nucleic acid hybridized; and

d) repeating steps a) to c) a sufficient number of times to determine a sequence of the target nucleic acid.

178. The method of claim 177, wherein the one or more nucleic acid probes are immobilized in an array.

179. The method of claim 177, wherein, prior to step b), the hybridized target nucleic acid is ligated to the determinable variable sequence.

180. A method for analyzing biological macromolecules in a sample comprising:

exposing the sample to infrared radiation, so that one or more of the biological macromolecules are desorbed and ionized; and

analyzing the sample using matrix-assisted laser desorption/ionization mass spectrometry;

wherein the accuracy of mass determination by matrix-assisted laser desorption/ionization is in the range of about 10² to about 5×

10³ ppm.

181. The method of claim 180, wherein the accuracy of mass determination by matrix-assisted laser desorption/ionization is in the range of about 100 to about 500 ppm.

182. The method of claim 180, wherein the accuracy of mass determination by matrix-assisted laser desorption/ionization is in the range of about 1×

10³ to about 5×

10³ ppm.

183. The method of claim 180, wherein the molecular weight of the biological macromolecule is 150 kDa or less;

and

the precision of mass determination by matrix-assisted laser desorption/ionization is in the range of about 400-500 ppm.

184. The method of claim 180, wherein the molecular weight of the biological macromolecule is 150 kDa or less;

and

the precision of mass determination by matrix-assisted laser desorption/ionization is in the range of about 200-400 ppm.

185. The method of claim 180, wherein the molecular weight of the biological macromolecule exceeds 1 MDa (megadalton).

186. The method of claim 180, further comprising extracting ions of the ionized biological macromolecules from an ion source by delayed extraction, whereby enhanced mass remixture is achieved.

187. The method of claim 180, wherein the matrix is glycerol.

188. The method of claim 180, wherein the matrix is succinic acid.

189. The method of claim 180, wherein the biological macromolecule is a polypeptide greater than 50 kDa and the matrix-assisted laser desorption/ionization mass spectrometry is performed using a reflectron time-of-flight format.

190. The method of claim 180 that is a method for detecting the presence or absence of a nucleic acid in a sample, wherein:

the sample is mixed with a matrix to form a homogeneous mixture with the nucleic acid; and

the nucleic acid is detected if present in the sample.

191. The method of claim 190, wherein the sample is a biological sample.

192. The method of claim 190, wherein the nucleic acid comprises at least 2000 nucleotides.

193. The method of claim 190, wherein the nucleic acid comprises at least 280 nucleotides.

194. The method of claim 190, wherein the nucleic acid is DNA.

195. The method of claim 190, wherein the nucleic acid comprises at least 1200 nucleotides.

196. The method of claim 190, wherein the nucleic acid is RNA.

197. The method of claim 190, wherein the presence or absence of the nucleic acid is indicative of the presence or absence of a genetic disease.

198. The method of claim 190, wherein the presence or absence of the nucleic acid is indicative of the presence or absence of a birth defect.

199. The method of claim 190, wherein the presence or absence of the nucleic acid is indicative of the presence or absence of an infectious organism.

200. The method of claim 190, wherein the presence or absence of the nucleic acid is indicative of the identity of a subject.

201. The method of claim 190, wherein the matrix is a substituted or unsubstituted alcohol.

202. The method of claim 190, wherein the nucleic acid/matrix mixture is deposited onto fields of a chip array, arrays of pins or beads in pits of flat surfaces.

203. A method for determining the presence or absence of a target biological macromolecule in a sample, comprising:

analyzing the sample using infrared matrix-assisted laser desorption/ionization mass spectrometry and a liquid matrix;

wherein the target biological macromolecule is detected if present in the sample.

204. The method of claim 203, wherein the sample is a biological sample.

205. The method of claim 203, wherein the sample contains one or more biological macromolecules other than the target biological macromolecule.

206. The method of claim 203, wherein the target macromolecule is a biopolymer.

207. The method of claim 206, wherein the target biopolymer is a polypeptide.

208. The method of claim 206, wherein the target biological macromolecule is a nucleic acid.

209. The method of claim 206, wherein the target nucleic acid is a double-stranded nucleic acid or the target nucleic acid is a single-stranded nucleic acid or the target nucleic acid comprises double-stranded and single-stranded regions.

210. The method of claim 206, wherein the presence or absence of the target biological macromolecule is indicative of one or more of the following:

the presence or absence of a genetic disease or is indicative of the presence or absence of a birth defect;

the presence or absence of an infectious organism;

the identity of a subject.

211. The method of claim 203, wherein delayed ion extraction is used in the matrix-assisted laser desorption/ionization mass spectrometry.

212. The method of claim 203, wherein the biological macromolecule is selected from the group consisting of a carbohydrate, a nucleoprotein, a proteoglycan, lipids, nucleic acid analogs and a macromolecular complex.

213. The method of claim 203, wherein the target biological macromolecule is immobilized on a solid support.

214. The method of claim 213, wherein the target biological macromolecule is immobilized to the solid support via a cleavable linkage and/or a reversible linkage.

215. The method of claim 213, wherein the target biological macromolecule is cleaved from the support during the step of performing matrix-assisted laser desorption/ionization mass spectrometry.

216. The method of claim 213, wherein the support comprises one or more hydrophilic areas and each of the one or more areas is surrounded by a hydrophobic area or one or more hydrophobic areas and each of the one or more areas is surrounded by a hydrophilic area or is the terminus of a fiber optic cable.

217. The method of claim 209, wherein a target nucleic acid is immobilized by hybridization to a complementary capture nucleic acid molecule, which is immobilized to a solid support.

218. The method of claim 203, wherein the target biological macromolecule is conditioned prior to the step of performing matrix-assisted laser desorption/ionization mass spectrometry.

219. The method of claim 206, wherein the target biopolymer is conditioned by a method selected from the group consisting of ion exchange, phosphodiester backbone modification effected by cation exchange;

contact with an alkylating agent or trialkylsilyl chloride;

incorporation of at least one nucleotide that reduces sensitivity for depurination in the target nucleic acid;

incorporation of at least one mass modified nucleotide in the target nucleic acid; and

hybridization of a tag probe to a portion of a nucleic acid molecule that contains the target nucleic acid but is distinct from a target nucleic acid sequence.

220. The method of claim 207, wherein the target polypeptide is obtained by in vitro translation, or by in vitro transcription followed by translation, of a nucleic acid encoding the target polypeptide.

221. The method of claim 220, wherein the nucleic acid encoding the target polypeptide further comprises a nucleotide sequence encoding a second polypeptide.

222. The method of claim 207, wherein the target polypeptide comprises a tag.

223. The method of claim 203, wherein:

the liquid matrix is glycerol; and

the biological macromolecule is a nucleic acid with a mass in the range from about 1×

10⁴ Daltons to about 1×

10⁶ Daltons.

224. The method of claim 203, further comprising, prior to analyzing the sample using matrix-assisted laser desorption/ionization mass spectrometry, depositing the sample on a substrate.

225. The method of claim 224, wherein said depositing is performed with an automated liquid dispensing device.

226. The method of claim 203, wherein infrared matrix-assisted laser desorption/ionization mass spectrometry is performed at a temperature in the range of about −

80°

C. to 20°

C.