Methods and compositions for analyzing proteins

• US 20030013126A1
• Filed: 05/21/2002
• Published: 01/16/2003
• Est. Priority Date: 05/21/2001
• Status: Active Grant

• Current Assignee: Monogram Biosciences, Inc. (Laboratory Corporation Of America Holdings)
• Original Assignee: Monogram Biosciences, Inc. (Laboratory Corporation Of America Holdings)
• Inventors: Singh, Sharat, Salimi-Moosavi, Hossein, Hernandez, Vincent S., Kirakossian, Hrair, Tahir, Syed Hasan, Matray, Tracy J., Wallweber, Gerald J.
• Granted Patent: US 7,255,999 B2
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2317/40 characterized by post-translational modification
• CPC Class Codes: G01N 33/582 with fluorescent label
• 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

Abstract

Methods, compositions and kits are disclosed for determining one or more target polypeptides in a sample where the target polypeptides have undergone a post-translational modification. A mixture comprising the sample and a first reagent comprising a cleavage-inducing moiety and a first binding agent for a binding site on a target polypeptide is subjected to conditions under which binding of respective binding moieties occurs. The binding site is the result of post-translational modification activity involving the target polypeptide. The method may be employed to determine the target polypeptide itself. In another embodiment the presence and/or amount of the target polypeptide is related to the presence and/or amount and/or activity of an agent such as an enzyme involved in the post-translational modification of the target polypeptide. The interaction between the first binding agent and the binding site brings the cleavage-inducing moiety into close proximity to a cleavable moiety, which is associated with the polypeptide and is susceptible to cleavage only when in proximity to the cleavage-inducing moiety. In this way, an electrophoretic tag for each of the polypeptides may be released. Released electrophoretic tags are separated and the presence and/or amount of the target polypeptides are determined based on the corresponding electrophoretic tags.

Claims

1. A method for determining the presence or absence of one or more target polypeptides in a sample suspected of containing the target polypeptides, the method comprising the steps of:

providing a class-specific reagent and one or more electrophoretic probes, the class-specific reagent having a cleavage-inducing moiety having an effective proximity and a first binding agent specific for a post-translational modification on the one or more target polypeptides, and the one or more electrophoretic probes each having a binding moiety specific for a target polypeptide and one or more electrophoretic tags each attached thereto by a cleavable linkage;

mixing the sample, the class-specific reagent, and the one or more electrophoretic probes such that the class-specific reagent and the one or more electrophoretic probes bind to the one or more target polypeptides and at least one cleavable linkage of the one or more electrophoretic probes is within the effective proximity of a cleavage-inducing moiety so that one or more electrophoretic tags are released;

electrophoretically separating the released electrophoretic tags; and

determining the presence or absence of the one or more target polypeptides based on the presence or absence of the released electrophoretic tags.

2. The method of claim 1 wherein said one or more target polypeptides is a plurality of target polypeptides.

3. The method of claim 2 wherein said cleavage-inducing moiety is a sensitizer that produces an active species.

4. The method of claim 3 wherein said sensitizer is a photosensitizer, said cleavable linkage is an oxidation-labile linkage, and said said active species is selected from the group consisting of hydrogen peroxide, hydroxyl radical, superoxide anion, phenoxy radical, and singlet oxygen.

5. The method of claim 4 wherein said active species is singlet oxygen.

6. The method of claim 5 wherein said binding agent of said class-specific reagent is selected from the group consisting of antibodies, protein receptors, ligands of protein receptors, lectins, biotin-containing moieties, boronic acid-containing moieties, aptamers, enzyme substrates, enzyme cofactors, and enzyme subunits, and wherein said binding moiety of said electrophoretic probe is an antibody or an antibody binding composition.

7. The method of claim 5 wherein said post-translational modification is selected from the group consisting of phosphorylation, glycosylation, ribosylation, acetylation, acylation, methylation, lipidation, isoprenylation and ubiquitination.

8. The method of claim 5 wherein said plurality of target polypeptides is in the range of from 2 to 100.

9. The method of claim 8 wherein said plurality of target polypeptides is in the range of from 5 to 50.

10. The method of claim 5 wherein said photosensitizer is selected from the group consisting of porphyrins, phthalocyanines, halogenated derivatives of a fluorescein dye, halogenated derivatives of a rhodamine dye, and naphthalocyanines.

11. The method of claim 5 wherein said cleavable linkage comprises an olefin, a thioether, a selenoether, a thiazole, an oxazole, or an imidazole.

12. The method of claim 5 wherein said electrophoretic probe is defined by the following formula:

T—

(L—

E)_k

wherein;

T is a binding moiety specific for a target polypeptide, L is an oxidation-labile linkage, E is an electrophoretic tag, and k is an integer greater than or equal to 1.

13. The method of claim 12 wherein L is selected from the group consisting of olefins, thioethers, selenoethers, thiazoles, oxazoles, and imidazoles.

14. The method of claim 13 wherein E is a fluorescent, water-soluble organic compound having a molecular weight in the range of from about 150 to 2500 daltons.

15. The method of claim 13 wherein E has the form (M,D), wherein D is a fluorescent dye and wherein M is a mobility modifying moiety that is a bond or an organic molecule having up to 100 atoms other than hydrogen selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur.

16. The method of claim 12 wherein said electrophoretic tag, E, is defined by the formula:

A—

M—

D

wherein;

A is —

C(═

O)R, where R is aliphatic, aromatic, alicyclic or heterocyclic having from 1 to 8 carbon atoms and 0 to 4 heteroatoms selected from the group consisting of O, S. and N;

—

CH₂—

C(═

O)—

NH—

CHO;

—

SO₂H;

—

CH₂—

C(═

O)O—

CHO;

—

C(═

O)NH—

(CH₂)_n—

NH—

C(═

O)C(═

O)—

(C₆H₅), where n is in the range of from 2 to 12;

D is a fluorescent dye; and

M is a bond or an organic molecule having up to 100 atoms other than hydrogen selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur, with the proviso that the total molecular weight of E be within the range of from about 150 to about 5000 daltons.

17. The method of claim 16 wherein D is a fluorescein dye.

19. The kit of claim 18 wherein said cleavage-inducing moiety is a photosensitizer and said cleavable linkage is an oxidation-labile linkage.

20. The kit of claim 19 wherein said first binding agent is selected from the group consisting of antibody binding compositions, protein receptors, ligands of protein receptors, lectins, biotin-containing moieties, boronic acid-containing moieties, aptamers, enzyme substrates, enzyme cofactors, and enzyme subunits.

21. The kit of claim 20 wherein said binding moieties of said one or more electrophoretic probes are antibody binding compositions.

22. The kit of claim 21 wherein said one or more electrophoretic probes are a plurality in the range of from 2 to 50.

23. The kit of claim 22 wherein each different electrophoretic probe is specific for a different target polypeptide.

24. The kit of claim 23 wherein each of said one or more electrophoretic probes is defined by the formula:

T—

(L—

E)_k

wherein;

T is said binding moiety specific for a target polypeptide, L is said oxidation-labile linkage, E is an electrophoretic tag, and k is an integer greater than or equal to 1.

26. The composition of claim 25 wherein L is an oxidation-labile linkage, said plurality is in the range of from 2 to 500, and E has a molecular weight in the range of from 150 to 10,000 daltons.

27. The composition of claim 26 wherein said electrophoretic probe is defined by the formula:

T—

(L—

(M, D))_k

wherein T, L, and k are defined as above;

D is a detection moiety; and

M is a bond or a water soluble organic compound consisting of from 1 to 100 atoms, not including hydrogen, that are selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur.

28. The composition of claim 27 wherein D is a fluorescent label, a chromogenic label, or an electrochemical label.

29. The composition of claim 28 wherein M is a polymer selected from any one of polyethers, polyesters, polypeptides, oligosaccharides, polyurethanes, polyamides, polysulfonamides, polysulfoxides, polyphosphonates, and block copolymers thereof.

30. The composition of claim 29 wherein D is a fluorescein.

31. The composition of claim 30 wherein said fluorescein is selected from the group consisting of 5- and 6-carboxyfluorescein, 5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dimethoxy-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dimethoxy-4′

,5′

-dichloro-5- and 6-carboxyfluorescein, 2′

,7′

-dimethoxy-4′

,5′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 1′

,2′

,7′

,8′

-dibenzo-5- and 6-carboxy-4,7-dichlorofluorescein, 1′

,2′

,7′

,8′

-dibenzo-4′

,5′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, and 2′

,4′

,5′

,7′

-tetrachloro-5- and 6-carboxy-4,7-dichlorofluorescein.

32. The composition of claim 27 wherein L is selected from the group consisting of olefins, thioethers, selenoethers, thiazoles, oxazoles, and imidazoles.

33. The composition of claim 27 wherein D is D₁-g-D₂, wherein one of D1 and D2 is an energy transfer acceptor molecule and the other of D₁ and D₂ is an energy transfer donor molecule, and wherein g is a rigid linker having a length selected so that energy transfer takes place between D₁ and D₂.

34. The composition of claim 33 wherein D₁ and D₂ are selected from a group consisting of rhodamines, fluoresceins, and halogenated derivatives thereof.

35. The composition of claim 27 wherein T is an antibody binding composition.

36. The composition of claim 35 wherein T is an antibody.

37. The composition of claim 27 wherein M is made by coupling a plurality of from 2 to 10 of the compounds selected from the group consisting of dimethoxytrityl-protected hexaethylene glycol phosphoramidite, 6-(4-Monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite, 12-(4-Monomethoxytritylamino)dodecyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite, 2-[2-(4-Monomethoxytrityl)aminoethoxy]ethyl-(2-cyanoethyl), N,N-diisopropyl)-phosphoramidite, (S-Trityl-6-mercaptohexyl)- (2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite, 9-O-Dimethoxytrityl-triethylene glycol, 1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 3(4,4′

Dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5′

-O-Dimethoxytrityl-1′

,2′

-Dideoxyribose-3′

-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 18-O Dimethoxytritylhexaethyleneglycol, 1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 12-(4,4′

-Dimethoxytrityloxy)dodecyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 1,3-bis-[5-(4,4′

-dimethoxytrityloxy)pentylamido]propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 1-[5-(4,4′

-dimethoxytrityloxy)pentylamido]-3-[5-fluorenomethoxycarbonyloxy pentylamido]-propyl-2-[(2-eyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, Tris-2,2,2-[3-(4,4′

-dimethoxytrityloxy)propyloxymethyl]ethyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, succinimidyl trans-4-(maleimidylmethyl) cyclohexane-1-carboxylate, succinimidyl 3-(2-pyridyldithio)propionate, succinimidyl acetylthioacetate, succinimidyl 4-(p-maleimidophenyl)butyrate;

N-γ

-maleimidobutyryl-oxysuccinimide ester;

p-nitrophenyl iodoacetate; and

4-(4-N-maleimidophenyl)butyric acid hydrazide.

38. The composition of claim 27 wherein said plurality of said electrophoretic probes is in the range of from 5 to 100.

39. The composition of claim 38 wherein said plurality of said electrophoretic probes is in the range of from 10 to 30.

18. A kit for detecting the presence or absence of a plurality of target polypeptides in a sample, the kit comprising:

a class-specific reagent having a cleavage-inducing moiety and a first binding agent specific for a post-translational modification on the one or more target polypeptides; and

one or more electrophoretic probes each having a binding moiety specific for a target polypeptide and one of more electrophoretic tags attached by a cleavable linkage.

25. A composition of matter for detecting one or more target polypeptides, the composition comprising a plurality of electrophoretic probes defined by the formula:

T—

(L—

E)_k

wherein;

T is a binding moiety specific for a target polypeptide, L is a cleavable linkage, E is an electrophoretic tag, and k is an integer greater than or equal to 1, such that each different T of the plurality is attached to a different E, and such that each E of the plurality has optical characteristics and/or an electrophoretic mobility that is distinct from those of every other E of the plurality.

40. A composition of matter for detecting the presence or absence of a target molecule in a sample, the compositon comprising a plurality of electrophoretic tags defined by the formula:

A—

M—

D

wherein;

A is —

C(═

O)R, where R is aliphatic, aromatic, alicyclic or heterocyclic having from 1 to 8 carbon atoms and 0 to 4 heteroatoms selected from the group consisting of O, S. and N;

—

CH₂-—

C(═

O)—

NH—

CHO;

—

SO₂H;

—

CH₂—

C(═

O)O—

CHO;

—

C(═

O)NH—

(CH₂)_n—

NH—

C(═

O)C(═

O)—

(C₆H₅), where n is in the range of from ₂ to 12;

D is a detection moiety; and

M is a bond or an organic molecule having up to 100 atoms other than hydrogen selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur, with the proviso that the total molecular weight of A—

M—

D be within the range of from about 150 to about 5000 daltons.

41. The composition of claim 40 wherein D is a fluorescent label, a chromogenic label, or an electrochemical label.

42. The composition of claim 41 wherein D is a fluorescent label.

43. The composition of claim 42 wherein D is a fluorescein.

44. The composition of claim 42 wherein D is D₁-g-D₂, wherein one of D1 and D2 is an energy transfer acceptor molecule and the other of D₁ and D₂ is an energy transfer donor molecule, and wherein g is a rigid linker having a length selected so that energy transfer takes place between D₁ and D₂.

45. The composition of claim 44 wherein D₁ and D₂ are selected from a group consisting of rhodamines, fluoresceins, and halogenated derivatives thereof.

47. The composition of claim 40 wherein D is a fluorescent label and wherein said plurality of said electrophoretic tags is in the range of from 5 to 100.

48. The composition of claim 47 wherein each electrophoretic tag of said plurality has a distinct charge-mass ratio or distinct optical characteristics so that each electrophoretic tag forms a detectable peak upon electrophoretic separation.

49. The composition of claim 48 wherein D is the same for each electrophoretic tag of said plurality and wherein each electrophoretic tag of said plurality has a distinct charge-mass ratio so that each electrophoretic tag forms a distinct peak upon electrophoretic separation.

50. The composition of claim 40 wherein M is a polymer selected from any one of polyethers, polyesters, polypeptides, oligosaccharides, polyurethanes, polyamides, polysulfonamides, polysulfoxides, polyphosphonates, and block copolymers thereof.

51. The composition of claim 50 wherein D is a fluorescein.

52. The composition of claim 51 wherein said fluorescein is selected from the group consisting of 5- and 6-carboxyfluorescein, 5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dimethoxy-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dimethoxy-4′

,5′

-dichloro-5- and 6-carboxyfluorescein, 2′

,7′

-dimethoxy-4′

,5′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 1′

,2′

,7′

,8′

-dibenzo-5- and 6-carboxy-4,7-dichlorofluorescein, 1′

,2′

,7′

,8′

-dibenzo-4′

,5′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 2′

,7′

-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, and 2′

,4′

,5′

,7′

-tetrachloro-5- and 6-carboxy-4,7-dichlorofluorescein.

53. A composition for attaching an electrophoretic tag to a binding moiety by a thioether cleavable linkage, the composition defined by the formula:

HOOC—

R₁—

S—

CH(R₄)—

C(═

O)—

NH—

R₂—

NH—

R₃

wherein R₁ is C₁-C₁₀ alkyldiyl;

R₂ is C₃-C₁₀ alkyldiyl;

R₃ is an amino protection group; and

R₄ is phenyl or electron-donating-substituted phenyl.

54. The composition of claim 53 wherein said amino protection group is Fmoc and R₄ is phenyl.

55. A composition comprising:

(a) a plurality of electrophoretic probes each comprising an electrophoretic tag attached thereto by a cleavable linkage capable of reacting with singlet oxygen; and

(b) a class-specific reagent comprising a photosensitizer, wherein the photosensitizer is capable, in its excited state, of activating oxygen to its singlet state.

56. The composition of claim 55 wherein said class-specific reagent comprises one or more photosensitizer beads.

57. The composition of claim 56 wherein said one or more photosensitizer beads each have a first binding agent attached.

58. The composition of claim 55 wherein said cleavable linkage contains an olefin group and one or more electron donating substituents in conjugation with said olefin group.

59. The composition of claim 55 wherein said plurality of electrophoretic probes is defined by the formula:

T—

(L—

E)_k

wherein;

T is a binding moiety specific for a target molecule, L is a cleavable linkage, E is an electrophoretic tag, and k is an integer greater than or equal to 1, such that each different T of said plurality is attached to a different E, and such that each E of the plurality has optical characteristics and/or an electrophoretic mobility that is distinct from those of every other E of said plurality.

60. The composition of claim 55 wherein said class-specific reagent specifically binds phosphorylated polypeptides.

61. A composition comprising:

(a) a target polypeptide;

(b) an electrophoretic probe specifically bound to the target polypeptide; and

(c) a class-specific reagent compising a cleavage-inducing moiety having an effective proximity, the class-specific reagent being specifically bound to the target polypeptide such that the electrophoretic probe is within said effective proximity.

62. The composition of claim 61 wherein said target polypeptide has a phosphoryl group and wherein said class-specific reagent is specifically bound thereto.