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 - 40 . (canceled) 
     
     
         41 . A method of isolating a modified protein from a spent cell culture medium, the method comprising:
 incubating a cell in a nutrient medium comprising an unnatural sugar which comprises a chemical handle;   allowing the unnatural sugar to be added onto a protein by glycosylation, thereby forming the modified protein;   allowing the cell to secrete the modified protein;   harvesting the spent medium comprising the modified protein; and,   isolating the modified secreted protein based on the presence of the chemical handle.   
     
     
         42 . The method of  claim 41  wherein the chemical handle comprises an alkyne reactive moiety. 
     
     
         43 . The method of  claim 42  wherein the alkyne reactive moiety is an azide. 
     
     
         44 . The method of  claim 43  wherein the azide comprises a GalNAz, ManNAz, GlcNAz, Ac 4 GalNAz, Ac 4 ManNAz, or a Ac 4 GlcNAz. 
     
     
         45 . The method of  claim 41 , wherein the cell comprises a eukaryotic cell, a mammalian cell, a hybridoma cell or a cell producing a recombinant protein. 
     
     
         46 . The method of  claim 45 , wherein the mammalian cell comprises a murine cell, a plant cell, a goat cell, a rabbit cell, or a chinese hamster ovary (CHO) cell. 
     
     
         47 . The method of  claim 41 , wherein the modified protein is isolated on an activated solid or semi-solid support that comprises a group that reacts with the chemical handle. 
     
     
         48 . The method of  claim 47 , wherein the activated solid or semi-solid support comprises an alkyne, phosphine or a cyclooctyne group. 
     
     
         49 . The method of  claim 47 , wherein the solid or semi-solid support comprises an agarose, an aerogel, a hydrogel, a resin, a bead, a biochip, a microfluidic chip, a silicon chip, a multi-well plate, a membrane, a conducting metal, a nonconducting metal, a glass, a magnetic support, a silica gel, a polymeric membrane, a particle, a derivatized plastic film, a glass bead, cotton, a plastic bead, an alumina gel, a polysaccharide, a poly(acrylate), a polystyrene, a poly(acrylamide), a polyol, an agar, a cellulose, a dextran, a starch, a FICOLL, a heparin, a glycogen, an amylopectin, a mannan, an inulin, a nitrocellulose, a diazocellulose, a polyvinylchloride, a polypropylene, a polyethylene, a nylon, a latex bead, a magnetic bead, a paramagnetic bead, a superparamagnetic bead, or a starch. 
     
     
         50 . The method of  claim 41  further comprising:
 comparing a total number of modified proteins in the spent medium based on the presence of the chemical handle, with a total number of secreted proteins in a second spent medium without the unnatural sugar, wherein the second spent medium is obtained from a second cell cultured in a second nutrient medium in the absence of the unnatural sugar. 
 
     
     
         51 . A method for increasing the concentration of a modified protein comprised within a spent cell culture medium, the method comprising:
 passing the spent cell culture medium over an activated solid or semi-solid matrix to immobilize the modified protein on the matrix;   washing the immobilized modified protein under stringent conditions;   treating the immobilized modified protein with a digestive enzyme to release the immobilized modified protein from the matrix,   wherein the activated solid or semi-solid matrix comprises a group that reacts with the chemical handle on the modified protein.   
     
     
         52 . The method of  claim 51  wherein the modified protein comprises an unnatural sugar comprising the chemical handle. 
     
     
         53 . The method of  claim 52  wherein the chemical handle comprises an alkyne reactive moiety. 
     
     
         54 . The method of  claim 53  wherein the alkyne reactive moiety is an azide. 
     
     
         55 . The method of  claim 54  wherein the azide comprises a GalNAz, ManNAz, GlcNAz, Ac 4 GalNAz, Ac 4 ManNAz, or Ac 4 GlcNAz. 
     
     
         56 . The method of  claim 51 , wherein the spent cell culture medium is obtained from culturing a cell that comprises a eukaryotic cell, a mammalian cell, a hybridoma cell or a cell producing a recombinant protein. 
     
     
         57 . The method of  claim 56 , wherein the mammalian cell comprises a murine cell, a plant cell, a goat cell, a rabbit cell, or a chinese hamster ovary (CHO) cell. 
     
     
         58 . The method of  claim 51 , wherein the activated solid or semi-solid matrix comprises an alkyne, phosphine or a cyclooctyne group. 
     
     
         59 . The method of  claim 51 , wherein the modified solid or semi-solid matrix comprises an agarose, an aerogel, a hydrogel, a resin, a bead, a biochip, a microfluidic chip, a silicon chip, a multi-well plate, a membrane, a conducting metal, a nonconducting metal, a glass, a magnetic support, a silica gel, a polymeric membrane, a particle, a derivatized plastic film, a glass bead, cotton, a plastic bead, an alumina gel, a polysaccharide, a poly(acrylate), a polystyrene, a poly(acrylamide), a polyol, an agar, a cellulose, a dextran, a starch, a FICOLL, a heparin, a glycogen, an amylopectin, a mannan, an inulin, a nitrocellulose, a diazocellulose, a polyvinylchloride, a polypropylene, a polyethylene, a nylon, a latex bead, a magnetic bead, a paramagnetic bead, a superparamagnetic bead, a slide, an array, an agarose gel, a polyacrylamide gel, a polymeric particle or glass or a starch.