Methods and compositions for analyzing 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.

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.   
   
   
       2 . The kit of  claim 1  wherein said cleavage-inducing moiety is a photosensitizer and said cleavable linkage is an oxidation-labile linkage. 
   
   
       3 . The kit of  claim 2  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. 
   
   
       4 . The kit of  claim 3  wherein said binding moieties of said one or more electrophoretic probes are antibody binding compositions. 
   
   
       5 . The kit of  claim 4  wherein said one or more electrophoretic probes are a plurality in the range of from 2 to 50. 
   
   
       6 . The kit of  claim 5  wherein each different electrophoretic probe is specific for a different target polypeptide. 
   
   
       7 . The kit of  claim 6  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. 
   
   
       8 . 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. 
   
   
       9 . The composition of  claim 8  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. 
   
   
       10 . The composition of  claim 9  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. 
   
   
       11 . The composition of  claim 10  wherein D is a fluorescent label, a chromogenic label, or an electrochemical label. 
   
   
       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. 
   
   
       13 . The composition of  claim 12  wherein D is a fluorescein. 
   
   
       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. 
   
   
       15 . The composition of  claim 10  wherein L is selected from the group consisting of olefins, thioethers, selenoethers, thiazoles, oxazoles, and imidazoles. 
   
   
       16 . The composition of  claim 10  wherein D is D 1 -g-D 2 , wherein one of D1 and D2 is an energy transfer acceptor molecule and the other of D 1  and D 2  is an energy transfer donor molecule, and wherein g is a rigid linker having a length selected so that energy transfer takes place between D 1  and D 2 . 
   
   
       17 . The composition of  claim 16  wherein D 1  and D 2  are selected from a group consisting of rhodamines, fluoresceins, and halogenated derivatives thereof. 
   
   
       18 . The composition of  claim 10  wherein T is an antibody binding composition. 
   
   
       19 . The composition of  claim 18  wherein T is an antibody. 
   
   
       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. 
   
   
       21 . The composition of  claim 10  wherein said plurality of said electrophoretic probes is in the range of from 5 to 100. 
   
   
       22 . The composition of  claim 21  wherein said plurality of said electrophoretic probes is in the range of from 10 to 30. 
   
   
       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:
   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; —CH 2 —C(═O)—NH—CHO; —SO 2 H; —CH 2 —C(═O)O—CHO; —C(═O)NH—(CH 2 ) n  NH—C(═O)C(═O)—(C 6 H 5 ), 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. 
 
   
   
       24 . The composition of  claim 23  wherein D is a fluorescent label, a chromogenic label, or an electrochemical label. 
   
   
       25 . The composition of  claim 24  wherein D is a fluorescent label. 
   
   
       26 . The composition of  claim 25  wherein D is a fluorescein. 
   
   
       27 . The composition of  claim 25  wherein D is D 1 -g-D 2 , wherein one of D1 and D2 is an energy transfer acceptor molecule and the other of D 1  and D 2  is an energy transfer donor molecule, and wherein g is a rigid linker having a length selected so that energy transfer takes place between D 1  and D 2 . 
   
   
       28 . The composition of  claim 27  wherein D 1  and D 2  are selected from a group consisting of rhodamines, fluoresceins, and halogenated derivatives thereof. 
   
   
       29 . The composition of  claim 23  wherein D is a fluorescent label and wherein said plurality of said electrophoretic tags is in the range of from 5 to 100. 
   
   
       30 . The composition of  claim 29  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. 
   
   
       31 . The composition of  claim 30  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. 
   
   
       32 . The composition of  claim 23  wherein M is a polymer selected from any one of polyethers, polyesters, polypeptides, oligosaccharides, polyurethanes, polyamides, polysulfonamides, polysulfoxides, polyphosphonates, and block copolymers thereof. 
   
   
       33 . The composition of  claim 32  wherein D is a fluorescein. 
   
   
       34 . The composition of  claim 33  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. 
   
   
       35 . A composition for attaching an electrophoretic tag to a binding moiety by a thioether cleavable linkage, the composition defined by the formula:
   HOOC—R 1 —S—CH(R 4 )—C(═O)—NH—R 2 —NH—R 3      
     wherein R 1  is C 1 -C 10  alkyldiyl; R 2  is C 3 -C 10  alkyldiyl; R 3  is an amino protection group; and R 4  is phenyl or electron-donating-substituted phenyl. 
   
   
       36 . The composition of  claim 35  wherein said amino protection group is Fmoc and R 4  is phenyl. 
   
   
       37 . 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.   
   
   
       38 . The composition of  claim 37  wherein said class-specific reagent comprises one or more photosensitizer beads. 
   
   
       39 . The composition of  claim 38  wherein said one or more photosensitizer beads each have a first binding agent attached. 
   
   
       40 . The composition of  claim 37  wherein said cleavable linkage contains an olefin group and one or more electron donating substituents in conjugation with said olefin group. 
   
   
       41 . The composition of  claim 37  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. 
   
   
       42 . The composition of  claim 37  wherein said class-specific reagent specifically binds phosphorylated polypeptides. 
   
   
       43 . A composition comprising:
 (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.   
   
   
       44 . The composition of  claim 43  wherein said target polypeptide has a phosphoryl group and wherein said class-specific reagent is specifically bound thereto.