Labeling and detection of post translationally modified proteins

Abstract

Provided in certain embodiments are new methods for forming azido modified biomolecule conjugates of reporter molecules, carrier molecules or solid support. In other embodiments are provided methods for enzymatically labeling a biomolecules with an azide group.

1 - 11 . (canceled) 
     
     
         12 . A method for forming a post-translationally modified phosphoprotein conjugate, wherein the method comprises:
 a) contacting a protein with an alkyne-modified nucleotide, wherein the nucleotide has an alkyne moiety placed on a gamma phosphate of the nucleotide, in the presence of an enzyme that will transfer the alkyne-modified gamma phosphate to the protein to form an alkyne-modified phosphoprotein, and wherein the enzyme is a kinase that can phosphorylate a serine, threonine or tyrosine residue; and   b) contacting the alkyne-modified phosphoprotein with a reporter molecule, carrier molecule or solid support that comprises an azido moiety to form a phosphoprotein reporter molecule, carrier molecule or solid support conjugate.   
     
     
         13 . A method for forming a post-translationally modified phosphoprotein conjugate, wherein the method comprises:
 a) contacting a protein with an azido-modified nucleotide, wherein the nucleotide has an azide moiety placed on a gamma phosphate of the nucleotide, in the presence of an enzyme that will transfer the azido-modified gamma phosphate to the protein to form a azido-modified phosphoprotein, and wherein the enzyme is a kinase that can phosphorylate a serine, threonine or tyrosine residue; and   b) contacting the azido-modified phosphoprotein with a reporter molecule, carrier molecule or solid support that comprises an alkyne group to form a phosphoprotein reporter molecule, carrier molecule or solid support conjugate.   
     
     
         14 . A method for forming a phosphoprotein conjugate, wherein the method comprises:
 a) contacting a phosphoprotein with a base solution that removes phosphates from threonine and serine residues of the phosphorylated protein to form a protein comprising dehydroalanine or dehydroamino-2-butyric acid;   b) contacting the dehydroalanine or dehydroamino-2-butyric acid with a compound that comprises a thiol or amine group and an azide group or an alkyne group to form an azido or alkyne modified protein;   c) contacting the alkyne or azido modified phosphoprotein with a reporter molecule, carrier molecule or solid support that comprises an azido moiety to form a phosphoprotein reporter molecule, carrier molecule or solid support conjugate.   
     
     
         15 - 40 . (canceled) 
     
     
         41 . The method according to  claim 12 , wherein the alkyne-modified nucleotide comprises a terminal alkyne group or an activated alkyne group. 
     
     
         42 . The method according to  claim 41 , wherein the alkyne-modified nucleotide is an ATP-alkyne having the following structure: 
       
         
           
           
               
               
           
         
         wherein X is a linker selected from a single covalent bond or a series of stable covalent bonds incorporating 1-30 nonhydrogen atoms selected from the group consisting of C, N, O, S and P, optionally wherein the linker comprises —C(O)NH—, —C(O)O—, —NH—, —S—, or O—. 
       
     
     
         43 . The method according to  claim 13 , wherein the alkyne group is a terminal alkyne group or an activated alkyne group. 
     
     
         44 . The method according to  claim 13 , wherein the azido-modified nucleotide is an ATP-azide having the following structure: 
       
         
           
           
               
               
           
         
         wherein X is a linker selected from a single covalent bond or a series of stable covalent bonds incorporating 1-30 nonhydrogen atoms selected from the group consisting of C, N, O, S and P, optionally wherein the linker comprises —C(O)NH—, —C(O)O—, —NH—, —S—, or O—. 
       
     
     
         45 . The method according to  claim 12 , wherein the reporter molecule is a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme or a radioisotope. 
     
     
         46 . The method according to  claim 12 , wherein the reporter molecule comprises a fluorophore selected from a xanthene, a cyanine, a coumarin, a borapolyazaindacene, a pyrene, or an oxazine. 
     
     
         47 . The method according to  claim 12 , wherein the carrier molecule is an amino acid, a peptide, a protein, an antibody or fragment thereof, an IgG binding protein, a fluorescent protein, a polysaccharide, a dextran, agarose, a nucleotide, a nucleoside, an oligonucleotide, a nucleic acid, a hapten, an avidin or streptavidin, a biotin, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a tyramine, a synthetic polymer, a polymeric microparticle, a biological cell, a cellular component, an ion chelating moiety, an enzymatic substrate, a virus, or a non-biological particle. 
     
     
         48 . The method according to  claim 12 , wherein the solid support is Sepharose, poly(acylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, a microfluidic chip, a silicon chip, a microscope slide, a multi-well plate, silica gels, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or supraparamagnetic bead. 
     
     
         49 . The method according to  claim 13 , wherein the reporter molecule is a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme or a radioisotope. 
     
     
         50 . The method according to  claim 13 , wherein the reporter molecule comprises a fluorophore selected from a xanthene, a cyanine, a coumarin, a borapolyazaindacene, a pyrene, or an oxazine. 
     
     
         51 . The method according to  claim 13 , wherein the carrier molecule is an amino acid, a peptide, a protein, an antibody or fragment thereof, an IgG binding protein, a fluorescent protein, a polysaccharide, a dextran, agarose, a nucleotide, a nucleoside, an oligonucleotide, a nucleic acid, a hapten, an avidin or streptavidin, a biotin, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a tyramine, a synthetic polymer, a polymeric microparticle, a biological cell, a cellular component, an ion chelating moiety, an enzymatic substrate, a virus, or a non-biological particle. 
     
     
         52 . The method according to  claim 13 , wherein the solid support is Sepharose, poly(acylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, a microfluidic chip, a silicon chip, a microscope slide, a multi-well plate, silica gels, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or supraparamagnetic bead. 
     
     
         53 . The method according to  claim 14 , wherein the alkyne group is a terminal alkyne group or an activated alkyne group. 
     
     
         54 . The method according to  claim 14 , wherein the reporter molecule is a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme or a radioisotope. 
     
     
         55 . The method according to  claim 14 , wherein the reporter molecule comprises a fluorophore selected from a xanthene, a cyanine, a coumarin, a borapolyazaindacene, a pyrene, or an oxazine. 
     
     
         56 . The method according to  claim 14 , wherein the carrier molecule is an amino acid, a peptide, a protein, an antibody or fragment thereof, an IgG binding protein, a fluorescent protein, a polysaccharide, a dextran, agarose, a nucleotide, a nucleoside, an oligonucleotide, a nucleic acid, a hapten, an avidin or streptavidin, a biotin, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a tyramine, a synthetic polymer, a polymeric microparticle, a biological cell, a cellular component, an ion chelating moiety, an enzymatic substrate, a virus, or a non-biological particle. 
     
     
         57 . The method according to  claim 14 , wherein the solid support is Sepharose, poly(acylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, a microfluidic chip, a silicon chip, a microscope slide, a multi-well plate, silica gels, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or supraparamagnetic bead.