Capture compounds, collections thereof and methods for analyzing the proteome and complex compositions

• US 7,858,560 B2
• Filed: 07/16/2002
• Issued: 12/28/2010
• Est. Priority Date: 07/16/2001
• Status: Expired due to Fees

• Current Assignee: Caprotec Bioanalytics GmbH
• Original Assignee: Caprotec Bioanalytics GmbH
• Inventors: Siddiqi, Suhaib, Köster, Hubert, Little, Daniel P.
• Primary Examiner(s): Lundgren; Jeffrey S
• Application Number:
  US10/197,954
• Publication Number:
  US 20030119021A1
• Time in Patent Office: 3,087 Days
• Field of Search: None
• US Class Current: 506/13
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00581 Mass
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/0072 Organic compounds
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins
• 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

Abstract

Capture Collections of capture compounds and collections thereof and methods using the compounds collections for the analysis of biomolecules are provided. In particular, collections, compounds and methods are provided for analyzing complex protein mixtures, such as the proteome. The compounds in the collections are multifunctional reagents that provide for the separation and isolation of biomolecules in complex protein mixtures. In the collections, each and every compound in the collection, has the formula:

Z is a trityl derivative, each of m and n independently is an integer that is 1 to 100; X, the reactivity function, covalently binds to amino acid side chains of biomolecules; Y, the selectivity function, modulates binding of X to the amino acid side chains in biomolecules such that X binds to fewer biomolecules when the selectivity moiety Y is present than in its absence; and Q permits separation or immobilization of capture compounds in the collection. Automated systems for performing the methods also are provided.

Claims

1. A collection of capture compounds, comprising a plurality of different capture compounds, wherein:

each and every capture compound in the collection has the formula;

2. The collection of claim 1, wherein Q permits separation by arraying of the capture compounds on a solid support by binding to the surface or a molecule thereon.

3. The collection of claim 1 that includes at least fifty different capture compounds.

4. The collection of claim 2 that includes at least fifty different capture compounds.

5. The collection of claim 1 that includes at least 100 different capture compounds.

6. The collection of claim 2 that includes at least 100 different capture compounds.

7. The collection of claim 1, wherein Q permits immobilization of capture compounds at addressable loci on a solid support.

8. A solid support, comprising the collection of compounds of claim 1, wherein capture compounds of the collection are arrayed at addressable loci on the support.

9. A solid support, comprising the collection of compounds of claim 2, wherein capture compounds of the collection are arrayed at addressable loci on the support.

10. The collection of claim 1, wherein:

X is selected from among an active ester, an active halo moiety, an amino acid side chain-specific functional group and a metal complex; and

Y is a monovalent group selected from among straight or branched chain alkyl, straight or branched chain alkenyl, straight or branched chain alkynyl, cycloalkvl, cycloalkenyl, cycloalkynyl, heterocyclyl, straight or branched chain heterocyclylalkyl, straight or branched chain heterocyclylalkenyl, straight or branched chain heterocyclylalkynyl, aryl, straight or branched chain arylalkyl, straight or branched chain arylalkenyl, straight or branched chain arylalkynyl, heteroaryl, straight or branched chain heteroarylalkyl, straight or branched chain heteroarylalkenyl, straight or branched chain heteroarylalkynyl, halo, straight or branched chain haloalkyl, pseudohalo, azido, cyano, nitro, OR⁶⁰, NR⁶⁰R⁶¹, COOR⁶⁰, C(O)R⁶⁰, C(O)NR⁶⁰R⁶¹, S(O)_qR⁶⁰, S(O)_qOR⁶⁰, S(O)_qNR⁶⁰R⁶¹, NR⁶⁰C(O)R⁶¹,NR⁶⁰C(O)NR⁶⁰R⁶¹, NR⁶⁰S(O)_qR⁶⁰, SiR⁶⁰R⁶¹R⁶², P(R⁶⁰)₂P(O)(R⁶⁰)₂, P(O)(OR⁶⁰)₂, P(O)(OR⁶⁰)(R⁶¹) and P(O)NR⁶⁰R⁶¹, where q is an integer from 0 to 2; and

each R⁶⁰, R⁶¹, and R6² is independently hydrogen, straight or branched chain alkyl, straight or branched chain alkenyl, straight or branched chain alkynyl, aryl, straight or branched chain aralkyl, straight or branched chain aralkenyl, straight or branched chain aralkynyl, heteroaryl, straight or branched chain heteroaralkyl, straight or branched chain heteroaralkenyl, straight or branched chain heteroaralkynyl, heterocyclyl, straight or branched chain heterocyclylalkyl, straight or branched chain heterocyclylalkenyl or straight or branched chain heterocyclylalkynyl.

11. The collection of claim 1, wherein X is —

C(═

)O-Ph-pNO₂, —

C(═

)O—

C₆F₅, —

C(═

O)—

O—

(N-succinimidyl), —

OCH₂—

I, —

OCH₂—

Br, —

OCH₂—

Cl, —

C(O)CH₂I, —

C(O)CH₂Br or —

C(O)CH₂Cl.

12. The collection of claim 1, wherein X is selected from the group consisting of an active ester, an active halo moiety, an amino acid side chain-specific functional group and a metal complex.

13. The collection of claim 12, wherein X is an α

-halo ether, an α

-halo carbonyl group, maleimido, a gold complex, a mercury complex, an epoxide or an isothiocyanate.

14. The collection of claim 12, wherein X is —

C(═

O)O-Ph-pNO₂, —

C(═

O)O—

C₆F₅, —

C(═

O)—

O—

(N-succinimidyl),—

OCH₂—

I,—

OCH₂—

Br,—

OCH₂—

Cl, —

C(O)CH₂I, —

C(O)CH₂Br or —

C(O)CH₂Cl.

15. The collection of claim 1, wherein member compounds comprise a mass modifying tag.

16. The collection of claim 15, wherein the mass modifying tag is selected from (i)-(vii) as follows:

(i) the mass modifying tag has the formula −

X¹R¹⁰−

, wherein;

X¹ is a divalent group selected from —

O—

, —

O—

C(O)—

(CH₂)_y—

C(O)O—

, —

NH—

NH—

C(O)—

, —

C(O)—

NH—

, —

NH—

NH—

C(O)—

(CH₂)_y—

C(O)O—

, —

NH—

NH—

C(S)—

NH—

, —

O—

P(O-alkyl)—

O—

, —

O—

SO₂—

O—

, —

O—

C(O)—

CH₂—

S—

, —

S—

, —

NH—

NH— and

17. The collection of claim 1, further comprising biomolecules covalently bound to one or more capture compounds in the collection.

18. The collection of claim 17, wherein the biomolecules comprise proteins.

19. A method for analysis of biomolecules, comprising:

a) contacting a composition comprising a biomolecule with a collection of capture compounds of claim 1 to form capture compound-biomolecule complexes; and

b) identifying or detecting bound biomolecules.

20. The method of claim 19, wherein capture compounds in the collection further comprise a solubility group W that influences the solubility properties of the capture compound, wherein Z is tetravalent to accommodate W.

21. The method of claim 19, wherein the biomolecules are proteins.

22. The method of claim 19, wherein:

the capture compounds are in an addressable array; and

each locus in the array contains a different capture compound.

23. The method of claim 19, wherein identification comprises mass spectrometric analysis of the bound biomolecules.

24. The collection of claim 1, wherein the biomolecules are proteins.

25. The method of claim 23, wherein the biomolecules are proteins.

26. The method of claim 25, wherein the biomolecules bound to the capture compounds are treated with a protease prior to mass spectrometric analysis.

27. The method of claim 19, wherein each capture compound in the collection comprises the same X moiety but differs in the Y moiety.

28. The method of claim 19, wherein each capture compound in the collection comprises different Y and Q moieties.

29. The method of claim 19, wherein each capture compound in the collection comprises different X and Y moieties.

30. A method for separating protein conformers, comprising:

contacting a composition comprising a biomolecule with a collection of capture compounds of claim 1;

separating the members of the collection; and

identifying the bound proteins from the mixture, whereby each conformer has different binding specificity for members of the collection.

31. The method of claim 30, wherein identification is effected by mass spectrometry.

32. The method of claim 30, wherein at least one conformer is associated with a disease.

33. A method for reducing diversity of a complex mixture of biomolecules, comprising:

contacting the mixture with a collection of capture compounds of claim 1 to form capture compounds with bound biomolecules; and

after contacting,

separating capture compounds with bound biomolecules.

34. A method for identification of phenotype-specific biomolecules, comprising:

sorting cells from a single subject according to a predetermined phenotype to produce at least two separated sets of cells;

contacting mixtures of biomolecules from each set of sorted cells with a collection of capture compounds of claim 1; and

comparing the binding patterns of biomolecules from each set bound to capture compounds to identify biomolecules that differ for each set, thereby identifying phenotype-specific biomolecules.

35. The method of claim 34, wherein the cells are synchronized or frozen in a metabolic state before sorting and/or after sorting.

36. The method of claim 34, wherein the biomolecules comprise proteins.

37. The method of claim 34, wherein the bound biomolecules are identified by mass spectrometry.

38. The method of claim 34, wherein the phenotypes are diseased and healthy phenotypes.

39. The method of claim 38, wherein the diseased phenotype is a tumor and the healthy phenotype is non-tumor.

40. The method of claim 19, wherein:

each sorting function Q is an oligonucleotide that includes a single-stranded portion of length “

j”

for hybridizing to a complementary oligonucleotide, wherein j is at least 5 bases; and

the method further comprises;

hybridizing the capture compounds to a set of complementary oligonucleotides, which attached to a solid support, wherein the hybridizing is effected before or after the contacting step, thereby immobilizing the capture compounds or capture compound biomolecule complexes on the solid support.

41. The method of claim 40, wherein the single-stranded oligonucleotides or oligonucleotide analogs that are complementary to the Q moiety on the capture compounds are immobilized on a solid support.

42. The method of claim 41, wherein the single stranded oligonucleotides or oligonucleotide analogs that are complementary to the Q moiety comprise an addressable array.

43. The method of claim 19, wherein the collection comprises at least 50, 100, 500 or 1000 different capture compounds.

44. The method of claim 19, wherein the contacting step is performed in an aqueous medium and the biomolecules are hydrophilic.

45. The method of claim 19, wherein the contacting step is performed in a hydrophobic medium and the biomolecules are hydrophobic.

46. The method of claim 19, wherein identification or detection is effected by mass spectrometric analysis of the biomolecules bound to capture compounds.

47. The method of claim 46, wherein the mass spectrometric format is matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry.

48. The method of claim 19, wherein:

the sorting function Q permits arraying the compounds on a solid support; and

the method further comprises arraying the capture compounds on a solid support before, during or after the contacting step, wherein;

the resulting biomolecule-capture compound complexes are at discrete spots on a solid support.

49. The method of claim 48, wherein mass spectrometric analysis of the bound biomolecules comprises:

(i) addition of matrix to the biomolecule-capture compound complexes; and

(ii) spot-by-spot matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry.

50. The method of claim 19, further comprising:

chemical or enzymatic treatment of the biomolecule-capture compound complexes to remove or cleave portions thereof prior to mass spectrometric analysis.

51. The method of claim 23, wherein mass spectrometric analysis of the bound biomolecules, comprises:

addition of matrix to the biomolecules bound to capture compounds; and

(ii) matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry of biomolecules bound to capture compounds.

52. The method of claim 19, wherein the composition comprising a biomolecule is a cell lysate.

53. The method of claim 52, wherein the cells from which the lysate is produced are synchronized or frozen in a metabolic state.

54. A system for analysis of mixtures of biomolecules, comprising:

a collection of claim 1;

a computer programmed with instructions for controlling and directing analysis of biomolecules using the collections;

a mass spectrometer; and

software for analysis of data produced by the mass spectrometer.

55. The system of claim 54 that is an automated system.

56. The system of claim 54, further comprising a liquid chromatographic device.

57. A method of processing the mass spectrometric data produced by the method claim 23, comprising:

(a) subtracting any background;

(b) reducing noise;

(c) calibrating molecular weight; and

(d) refining peaks.

58. The method of claim 57, wherein step (d) comprises peak integration.

59. The method of claim 57, further comprising:

(e) comparing the processed data with existing protein databases or DNA databases containing open reading frames to determine whether the protein is known, and

(f) if the protein is known, identifying modifications.

60. The method of claim 57, further comprising:

comparing data from tissues of healthy and diseased individuals, or from different physiological or developmental stages, or from different parts of a tissue.

61. A method of analysis of biomolecules, comprising:

(a) reacting a first mixture of biomolecules with the collection of capture compounds of claim 1 to form a mixture of capture compound-bound biomolecules, wherein the capture compounds comprising the first mixture have a first mass modifying tag;

(b) reacting a second mixture of biomolecules with the collection of capture compounds of claim 1 to form a second mixture of capture compound-bound biomolecules, wherein the compounds comprising the second mixture of capture compound-bound molecules have either (i) no mass modifying tag;

or (ii) a second mass modifying tag;

(c) pooling the products of steps (a) and (b) to produce a mixture thereof;

(d) sorting the differently tagged capture compound-bound biomolecules in the mixture of step c) according to Q moiety to produce an array of sorted complexes; and

(e) analyzing the complexes at each locus.

62. The method of claim 23, wherein the analysis is orthogonal time of flight (O-TOF) mass spectrometry.

63. The method of claim 23, wherein the analysis is electrospray (ES) mass spectrometry.

64. A method for analyzing biomolecule interactions, comprising:

a) contacting a mixture of biomolecules with a collection of capture compounds of claim 1 to form capture compound-bound biomolecules, wherein the complexes are stable to matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry conditions;

b) contacting the capture compound-bound biomolecules with a mixture containing compounds selected from mixtures of biomolecules or mixtures of small molecules, wherein compounds in the mixture bind to biomolecules bound to capture compounds;

c) before or after step b) immobilizing the capture compounds on a solid support via the Q group of each set of capture compounds;

d) analyzing the bound compounds by mass spectrometry.

65. The method of claim 64, wherein the small molecules are candidate drugs and are selected from the group consisting of small organic molecules, peptides, peptide mimetics, antibodies, fragments of antibodies and recombinant or synthetic antibodies and fragments thereof;

and

the method is a method for identifying candidate drugs that bind to biomolecules.

66. The method of claim 64, wherein the capture compound-biomolecule complexes formed in step a) are contacted in step b) with a mixture of biomolecules to identify components of biomolecule complexes or biochemical pathways.

67. The method of claim 64, wherein the biomolecules are proteins.

68. The collection of claim 1, wherein the capture compounds further comprise a solubility group W that influences the solubility properties of the capture compound, wherein Z is tetravalent to accommodate W.

69. The collection of claim 1, wherein the selectivity function Y is selected from those set forth in FIGS. 17a-17hhhh and/or the reactivity function X is selected from those set forth in FIGS. 16a-b.

70. The collection of claim 1, wherein each capture compound in the collection comprises the same X moiety but differs in the Y moiety.

71. The collection of claim 1, wherein X is a photoreactive group.

72. The method of claim 64, wherein:

the mixtures of biomolecules comprise protein mixtures; and

the method is for determining biomolecule-protein or biomolecule-small molecule interactions.

73. The method of claim 19, wherein:

the biomolecule is a protein; and

the contacting step is performed under conditions whereby the interactions of the moiety Y of the capture compounds with protein in the composition are kinetically controlled.

74. The collection of claim 1, wherein Y is selected from among a receptor ligand, an enzyme substrate, an enzyme inhibitor, a transition state analog, a glue peptide, a peptide mimetic and a statin.

75. The collection of claim 1, wherein Y is a peptide ligand selected from among the peptide ligands set forth herein as SEQ ID NO:

1 through SEQ ID NO;

149.

76. The collection of claim 1, wherein Q comprises a fluorescent tag.

77. A collection of capture compounds, wherein each and every member of the collection has the formula:

78. The collection of claim 77, wherein X is C(═

O)O Ph pNO2, C(═

O)O C6F5, C(═

O) O (N-succinimidyl), OCH2 I, OCH2 Br, OCH2 Cl, C(O)CH2I, C(O)CH2Br or C(O)CH2Cl.