METHODS AND COMPOSITIONS FOR ANALYZING PROTEINS
First Claim
1. 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.
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Accused Products
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.
111 Citations
44 Claims
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1. A kit for detecting the presence or absence of a plurality of target polypeptides in a sample, the kit comprising:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A composition of matter for detecting one or more target polypeptides, the composition comprising a plurality of electrophoretic probes defined by the formula:
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T-(L-E)kwherein;
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.- View Dependent Claims (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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.
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11. The composition of claim 10 wherein D is a fluorescent label, a chromogenic label, or an electrochemical label.
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12. The composition of claim 11 wherein M is a polymer selected from any one of polyethers, polyesters, polypeptides, oligosaccharides, polyurethanes, polyamides, polysulfonamides, polysulfoxides, polyphosphonates, and block copolymers thereof.
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13. The composition of claim 12 wherein D is a fluorescein.
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14. The composition of claim 13 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.
- ,7′
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15. The composition of claim 10 wherein L is selected from the group consisting of olefins, thioethers, selenoethers, thiazoles, oxazoles, and imidazoles.
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16. The composition of claim 10 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.
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17. The composition of claim 16 wherein D1 and D2 are selected from a group consisting of rhodamines, fluoresceins, and halogenated derivatives thereof.
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18. The composition of claim 10 wherein T is an antibody binding composition.
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19. The composition of claim 18 wherein T is an antibody.
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20. The composition of claim 10 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, 18-0 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-cyanoethyl)-(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.
- Dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5′
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21. The composition of claim 10 wherein said plurality of said electrophoretic probes is in the range of from 5 to 100.
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22. The composition of claim 21 wherein said plurality of said electrophoretic probes is in the range of from 10 to 30.
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23. A composition of matter for detecting the presence or absence of a target molecule in a sample, the composition comprising a plurality of electrophoretic tags defined by the formula:
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A-M-Dwherein; 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. - View Dependent Claims (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
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35. A composition for attaching an electrophoretic tag to a binding moiety by a thioether cleavable linkage, the composition defined by the formula:
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HOOC—
R1—
S—
CH(R4)—
C(═
O)—
NH—
R2—
NH—
R3wherein 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.- View Dependent Claims (36)
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37. A composition comprising:
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(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. - View Dependent Claims (38, 39, 40, 41, 42)
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43. A composition comprising:
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(a) a target polypeptide; (b) an electrophoretic probe specifically bound to the target polypeptide; and (c) a class-specific reagent comprising 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. - View Dependent Claims (44)
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Specification