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 .- 16 . (canceled) 
     
     
         17 . A method of detecting an azido modified biomoelcule, comprising:
 a) forming an azide-alkyne cycloaddition reaction mixture comprising:
 a reporter molecule that comprises a terminal alkyne moiety: 
 an azido modified biomoelcule; 
 copper ions; 
 at least one reducing agent; and 
 a copper chelator; 
   b) incubating the azide-alkyne cycloaddition reaction mixture for a sufficient amount of time to form a biomoelcule-reporter molecule conjugate;   c) separating the biomoelcule-reporter molecule conjugate by size and/or weight of the glycoprotein-reporter molecule conjugate to form a separated biomoelcule-reporter molecule conjugate;   d) illuminating the separated biomoelcule-reporter molecule conjugate with an appropriate wavelength to form an illuminated biomoelcule-reporter molecule conjugate;   e) observing the illuminated biomolecule-reporter molecule conjugate wherein the glycoprotein is detected.   
     
     
         18 .- 39 . (canceled) 
     
     
         40 . A kit comprising:
 UDP-GalNAz;   a GalT enzyme;   an azide reactive reporter molecule, carrier molecule or solid support.   
     
     
         41 . A method of forming a glycoprotein conjugate, wherein the method comprises:
 a) contacting the glycoprotein with an endonuclease enzyme to form a deglycosylated glycoprotein;   b) contacting the deglycosylated glycoprotein with an azido modified carbohydrate in the presence of a galactosyltransferase enzyme to form an azido-modified glycoprotein; and   c) contacting the azido-modified glycoprotein with a reporter molecule, carrier molecule or solid support that comprises an azide reactive moiety to form the glycoprotein conjugate.   
     
     
         42 . The method according to  claim 41 , wherein the azide reactive moiety is an activated alkyne. 
     
     
         43 . The method according to  claim 41 , wherein the reporter molecule is a xanthene, cyanine, coumarin, borapolyazaindacene or pyrene dye. 
     
     
         42 . The method according to  claim 41 , wherein the reporter molecule is an enzyme substrate or hapten. 
     
     
         43 . The method according to  claim 41 , wherein the glycoprotein is an antibody. 
     
     
         44 . The method according to  claim 41 , wherein the carrier molecule is an amino acid, a peptide, a protein, a polysaccharide, a nucleotide, a nucleoside, an oligonucleotide, a nucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell or a virus. 
     
     
         45 . The method according to  claim 41 , wherein the carrier molecule comprises an antibody or fragment thereof, an avidin or streptavidin, a biotin, a blood component protein, a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG binding protein, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a microorganism, a metal binding protein, a metal chelating moiety, a non-biological microparticle, a peptide toxin, a phosphotidylserine-binding protein, a structural protein, a small-molecule drug, or a tyramide. 
     
     
         46 . The method according to  claim 41 , wherein the solid support is a microfluidic chip, a silicon chip, a microscope slide, a microplate well, 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 superparamagnetic bead. 
     
     
         47 . The method according to  claim 41 , wherein the solid support is Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose or starch.