Mass spectrometric detection of polypeptides

• US 6,322,970 B1
• Filed: 09/02/1998
• Issued: 11/27/2001
• Est. Priority Date: 09/02/1997
• Status: Expired due to Term

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Köster, Hubert, Little, Daniel, Higgins, G. Scott, Lough, David
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US09/146,054
• Time in Patent Office: 1,182 Days
• Field of Search: 435/4, 435/6, 435/69.1, 436/89
• US Class Current: 506/6
• CPC Class Codes: G01N 33/6818 Sequencing of polypeptides
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry
• CPC Class Codes: G01N 33/6851 Methods of protein analysis involving laser desorption ionisation mass spectrometry
• CPC Class Codes: H01J 49/00 Particle spectrometers or separator tubes

Abstract

A process for determining the identity of a target polypeptide using mass spectroscopy is provided. Depending on the target polypeptide to be identified, a process as disclosed can be used, for example, to diagnose a genetic disease or chromosomal abnormality, a predisposition to a disease or condition, or infection by a pathogenic organism; or for determining identity or heredity. Kits for performing the disclosed processes also are provided.

Claims

1. A process for obtaining information on a sequence of a target nucleic acid molecule by determining the identity of a polypeptide encoded by the nucleic acid molecule, comprising the steps of:

a) preparing the encoded polypeptide from a target nucleic acid molecule by in vitro translation, or by in vitro transcription followed by translation, of the target nucleic acid molecule;

b) determining the molecular mass of the encoded polypeptide by mass spectrometry; and

c) determining the identity of the polypeptide by comparing the molecular mass of the polypeptide with the molecular mass of a corresponding known polypeptide, thereby obtaining information on a sequence of nucleotides in the target nucleic acid molecule.

2. The process of claim 1, wherein the nucleic acid encoding the encoded polypeptide is RNA, and wherein the encoded polypeptide is obtained by in vitro translation.

3. The process of claim 1, wherein an RNA encoding the encoded polypeptide is prepared by in vitro transcription of the nucleic acid encoding the encoded polypeptide, and wherein the encoded polypeptide is obtained by in vitro translation of the RNA.

4. The process of claim 1, further comprising amplifying the nucleic acid encoding the encoded polypeptide.

5. The process of claim 4, wherein the amplifying is performed using a forward primer and a reverse primer.

6. The process of claim 4, wherein the amplifying is performed using a primer comprising a nucleotide sequence encoding a regulatory element selected from the group consisting of a ribosome binding site, a START codon and a transcription start signal, wherein, following amplification, the regulatory element is operably linked to the nucleic acid encoding the encoded polypeptide.

7. The process of claim 4, wherein the amplifying is performed using a primer comprising a nucleotide sequence encoding an RNA polymerase promoter, wherein, following amplification, the promoter is operably linked to the nucleic acid encoding the encoded polypeptide.

8. The process of claim 7, wherein the RNA polymerase promoter is selected from the group consisting of SP6 promoter, T3 promoter, and T7 promoter.

9. The process of claim 1, wherein the nucleic acid further comprises an operably linked exogenous nucleotide sequence encoding a regulatory element selected from the group consisting of an RNA polymerase promoter, a ribosome binding site, a START codon, and a transcription start signal.

10. The process of claim 1, wherein the nucleic acid comprises a nucleotide sequence, or complement thereof, encoding a second polypeptide.

11. The process of claim 10, wherein the second polypeptide is a tag peptide.

12. The process of claim 11, wherein the tag peptide is selected from the group consisting of a myc epitope, a Haemophilus influenza hemagglutinin peptide, a polyhistidine sequence, a polylysine sequence, a polyarginine sequence, and glutathione-S-transferase.

13. The process of claim 1, wherein the encoded polypeptide comprises a tag.

14. The process of claim 13, wherein the tag is biotin or a derivative thereof.

15. The process of claim 13, wherein the tag is a tag peptide, which is conjugated to the encoded polypeptide.

16. The process of claim 2, wherein the in vitro translation is performed in a cell-free extract.

17. The process of claim 16, wherein the cell-free extract is a eukaryotic cell-free extract.

18. The process of claim 17, wherein the eukaryotic cell-free extract is selected from the group consisting of a reticulocyte lysate, a wheat germ extract, and a combination thereof.

19. The process of claim 3, wherein the in vitro transcription is performed in a cell-free extract, and wherein translation of the target polypeptide is performed in the same cell-free extract.

20. The process of claim 19, wherein the cell-free extract comprises a reticulocyte lysate.

21. The process of claim 19, wherein the cell-free extract is a prokaryotic cell-free extract.

22. The process of claim 21, wherein the prokaryotic cell-free extract is an E. coli cell-free extract.

23. The process of claim 22, wherein the cell-free extract is E. coli S30 cell-free extract.

24. The process of claim 1, wherein transcription or translation is performed in vivo.

25. The process of claim 24, which is performed in a host cell.

26. The process of claim 25, wherein the host cell is a bacterium.

27. The process of claim 1, wherein the encoded polypeptide is isolated prior to mass spectrometry.

28. The process of claim 27, wherein the encoded polypeptide is isolated be reaction with an antibody.

29. The process of claim 13, wherein the encoded polypeptide is isolated by reaction with a reagent that interacts specifically with the tag.

30. The process of claim 29, wherein the tag is a tag peptide and the reagent is an antibody.

31. The process of claim 29, wherein the tag is a polyhistidine tag peptide and the reagent is a metal ion selected from the group consisting of nickel ions and cobalt ions, or wherein the tag is a polylysine or a polyarginine tag peptide and the reagent is selected from the group consisting of copper ions and zinc ions, wherein the reagent is chelated to a solid support.

32. The process of claim 29, wherein the tag is biotin or a derivative thereof and the reagent is selected from the group consisting of avidin and streptavidin.

33. The process of claim 1, wherein, prior to determining the molecular mass of the target polypeptide by mass spectrometry, the target polypeptide is immobilized on a solid support.

34. The process of claim 33, wherein the encoded polypeptide is immobilized to the solid support through a cleavable linker.

35. The process of claim 34, wherein the cleavable linker is selected from the group consisting of an acid cleavable linker, acid-labile linker, heat sensitive linker, and a photocleavable linker.

36. The process of claim 33, wherein the encoded polypeptide is immobilized by interacting specifically with a polypeptide that is conjugated to the solid support.

37. The process of claim 33, wherein the solid support is selected from the group consisting of a support having a flat surface and a support having a surface with a structure.

38. The process of claim 1, wherein the mass spectrometry is selected from the group consisting of matrix assisted laser desorption ionization (MALDI), delayed extraction MALDI, continuous or pulsed electrospray, ionspray, thermospray, or massive cluster impact and a detection format selected from the group consisting of linear time-of-flight, reflectron time-of-flight, single quadrupole, multiple quadrupole, single magnetic sector, multiple magnetic sector, Fourier transform ion cyclotron resonance, ion trap, and combinations thereof.

39. The process of claim 1, wherein the mass spectrometry is matrix-assisted laser desorption/ionization time-of-flight spectrometry.

40. The process of claim 1, wherein the encoded polypeptide is encoded by an allelic variant of a polymorphic region of a chromosome in a subject.

41. The process of claim 40, wherein the polymorphic region is in a gene.

42. The process of claim 40, wherein the polymorphic region is not in a gene.

43. The process of claim 40, wherein the allelic variant is associated with a disease or condition, thereby indicating that the subject has or is at risk of developing the disease or condition.

44. The process of claim 43, wherein the disease or condition is associated with an abnormal number of nucleotide repeats in the allelic variant.

45. The process of claim 44, wherein the nucleotide repeats are trinucleotide repeats.

46. The process of claim 45, wherein the disease or condition is selected from the group consisting of Huntington'"'"'s disease, prostate cancer, Fragile X syndrome type A, myotonic dystrophy type I, Kennedy disease, Machado-Joseph disease, dentatorubral and pallidolyusian atrophy, spino bulbar muscular atrophy and aging.

47. The process of claim 41, wherein the gene is selected from the group consisting of BRCA1, BRCA2, APC, dystrophin gene, β

-globin, Factor IX, Factor VIIc, ornithine-d-amino-transferase, hypoxanthine guanine phosphoribosyl transferase, CFTR, p53, and a proto-oncogene.

48. The process of claim 40, wherein the allelic variant is due to a point mutation.

49. The process of claim 41, wherein the polymorphic region is associated with graft rejection and the process is for determining compatibility between a donor and a recipient of a graft.

50. The process of claim 49, wherein the polymorphic region is the major histocompatibility locus.

51. The process of claim 40, wherein the encoded polypeptide is encoded by a nucleic acid comprising nucleotide repeats and the process is for a use selected from the group consisting of genotyping the subject, forensic analysis, and paternity testing.

52. The process of claim 51, wherein genotyping is performed by quantifying the number of nucleotide repeats.

53. The process of claim 51, wherein the nucleotide repeats are dinucleotide, trinucleotide, tetranucleotide, or pentanucleotide repeats.

54. The process of claim 42, wherein the gene is a mitochondrial gene.

55. The process of claim 1, wherein the encoded polypeptide is obtained from an infectious organism.

56. The process of claim 55, wherein the infectious organism is selected from the group consisting of a virus, a bacterium, a fungus, and a protist.

74. The process of claim 33, wherein the solid support is selected from the group consisting of beads, beads in an array of pits, capillaries, flat supports, plastic materials, wafers, combs, pins, arrays of pins, needles, arrays of needles, wells and nanoliter wells.

75. The process of claim 74, wherein the beads are selected from the group consisting of silica gel beads, controlled pore glass beads, magnetic beads, 4-(hydroxymethyl)phenoxymethylcopoly(styrene-1% divinylbenzene) resin, chloromethylated copolystyrene-divinylbenzene resin, cellulose beads, agarose beads and dextran beads.

76. The process of claim 74, wherein the flat supports are selected from the group consisting of glass fiber filters, glass surfaces, and metal surfaces.

77. The process of claim 76, wherein the metal surfaces are selected from the group consisting of steel, gold, silver, aluminum, silicon, and copper.

78. The process of claim 74, wherein the plastic materials are selected from the group consisting of polyethylene, polypropylene, polyamide, and polyvinylidene difluoride.

79. The process of claim 74, wherein the wafers are selected from the group consisting of silicon wafers, wafers with pits, wafers with filter bottoms, and wafers without filter bottoms.

80. The process of claim 33, wherein the target polypeptide is linked to the solid support by an interaction selected from the group consisting of hydrophobic interaction, hydrophilic interaction, and ionic interaction.

81. The process of claim 33, wherein the N-terminal amino acid of the target polypeptide is linked to the solid support.

82. The process of claim 33, wherein the C-terminal amino acid of the target polypeptide is linked to the solid support.

83. The process of claim 33, wherein the target polypeptide is covalently conjugated to the solid support.

84. The process of claim 83, wherein the target polypeptide is covalently conjugated to the solid support by reaction with a thiol-reactive functionality.

57. A process for obtaining information on sequences of a plurality of nucleic acid molecules by determining the identity of a plurality of target polypeptides encoded by the nucleic acid molecules, comprising the steps of:

a) obtaining a plurality of nucleic acid molecules encoding a plurality of target polypeptides;

b) preparing a plurality of differentially mass modified target polypeptides from the plurality of nucleic acid molecules;

c) determining the molecular mass of each differentially mass modified target polypeptide in the plurality by mass spectrometry; and

d) determining the identity of each target polypeptide in the plurality by comparing the molecular mass of each differentially mass modified target polypeptide in the plurality with the molecular mass of a corresponding known polypeptide, thereby obtaining information on nucleotide sequences in the plurality of nucleic acid molecules encoding the target polypeptides;

wherein each encoded polypeptide is immobilized to the solid support through a cleavable linker.

58. The process of claim 57, wherein the encoded polypeptides are obtained by in vitro translation, or by in vitro transcription, followed by translation, of a nucleic acid encoding the encoded polypeptide.

59. The process of claim 57, wherein, prior to determining the molecular mass of each differentially mass modified encoded polypeptide by mass spectrometry, each encoded polypeptide is immobilized on a solid support.

85. The process of claim 57, wherein each target polypeptide is conditioned prior to step c).

86. The process of claim 85, wherein the target polypeptide is immobilized to a solid support prior to conditioning.

87. The process of claim 85, wherein the conditioning is selected from the group consisting of anion exchange, cation exchange, treatment with an alkylating agent, treatment with trialkylsilylchloride, and incorporation of modified amino acids.

92. The process of claim 59, wherein the cleavable linker is selected from the group consisting of an acid cleavable linker, acid-labile linker, heat sensitive linker, and a photocleavable linker.

93. The process of claim 59, wherein the solid support is selected from the group consisting of a support having a flat surface and a support having a surface with a structure.

94. The process of claim 59, wherein each target polypeptide is immobilized in an array to the solid support.

95. The process of claim 59, wherein each target polypeptide is immobilized due to its interacting specifically with a polypeptide of interest, wherein the polypeptide of interest is conjugated in an array to the solid support.

60. A process for identifying a target nucleic acid molecule by determining the identity of a polypeptide encoded by the nucleic acid molecule, comprising the steps of:

a) preparing the encoded polypeptide by in vitro translation, or by in vitro transcription followed by translation, of a target nucleic acid molecule encoding the polypeptide;

b) contacting the encoded polypeptide with at least one agent that cleaves at least one peptide bond in the encoded polypeptide to produce peptide fragments of the encoded polypeptide;

c) determining the molecular mass of at least one of the peptide fragments of the encoded polypeptide by mass spectrometry; and

d) comparing the molecular mass of the peptide fragments of the encoded polypeptide with the molecular mass of peptide fragments of a corresponding known polypeptide, thereby determining the identity of the encoded polypeptide and the target nucleic acid molecule.

61. The process of claim 60, wherein the encoded polypeptide is immobilized to a solid support prior to contacting the encoded polypeptide with the agent.

62. The process of claim 60, wherein the encoded polypeptide is immobilized to the solid support through a cleavable linker.

63. The process of claim 62, wherein the encoded polypeptide is immobilized to the solid support through a chemically cleavable linker at one terminus of the polypeptide and through a photocleavable linker at the other terminus of the polypeptide.

64. The process of claim 60, wherein the encoded polypeptide is conditioned prior to step c), or the peptide fragments of the encoded polypeptide are conditioned prior to step d).

65. The process of claim 60, wherein the agent that cleaves at least one peptide bond in the encoded polypeptide is an endopeptidase.

88. The process of claim 64, wherein the target polypeptide is immobilized to a solid support prior to conditioning.

89. The process of claim 64, wherein the conditioning is selected from the group consisting of anion exchange, cation exchange, treatment with an alkylating agent, treatment with trialkylsilylchloride, and incorporation of modified amino acids.

91. The process of claim 64, wherein the conditioning is selected from the group consisting of anion exchange, cation exchange, treatment with an alkylating agent, treatment with trialkylsilylchloride, and incorporation of modified amino acids.

66. A process for obtaining information on sequences of a plurality of nucleic acid molecules by determining the identity of a plurality of encoded polypeptides encoded by the nucleic acid molecules, comprising the steps of:

a) obtaining a plurality of nucleic acid molecules encoding a plurality of encoded polypeptides;

b) preparing a plurality of encoded polypeptides from the plurality of nucleic acid molecules;

c) contacting each encoded polypeptide with at least one agent that cleaves at least one peptide bond in each encoded polypeptide to produce peptide fragments of each encoded polypeptide;

d) determining the molecular mass of at least one of the peptide fragments of each encoded polypeptide in the plurality by mass spectrometry; and

e) determining the identity of each encoded polypeptide in the plurality by comparing the molecular mass of the peptide fragments of each encoded polypeptide with the molecular mass of peptide fragments of a corresponding known polypeptide, thereby obtaining information on nucleotide sequences in the plurality of nucleic acid molecules encoding the encoded polypeptides.

67. The process of claim 66, wherein each encoded polypeptide is mass modified prior to step b), or the at least one peptide fragment of each encoded polypeptide is mass modified prior to step c).

68. The process of claim 66, wherein each encoded polypeptide in the plurality is immobilized to a solid support prior to contacting each encoded polypeptide with the agent.

69. The process of claim 66, wherein each encoded polypeptide is immobilized to the solid support through a cleavable linker.

70. The process of claim 66, wherein each encoded polypeptide is conditioned prior to step c), or the at least one peptide fragment of each encoded polypeptide is conditioned prior to step d).

71. (Amended) The process of claim 68, wherein the plurality of encoded polypeptides are immobilized in an array.

72. The process of claim 66, wherein the agent that cleaves at least one peptide bond in each encoded polypeptide is an endopeptidase.

73. The process of claim 66, wherein each encoded polypeptide is immobilized to the solid support through an chemically cleavable linker at one terminus of the polypeptide and through a photocleavable linker at the other terminus of the polypeptide.

90. The process of claim 70, wherein the target polypeptide is immobilized to a solid support prior to conditioning.