Method for analyzing a glycomolecule

Abstract

The invention relates generally the structural analysis of glycomolecule-containing macromolecules, such as those that occur either attached to proteins (proteoglycans, glycoproteins), lipids, or as free saccharides.

1 . A method for determining a glycomolecule fingerprint for a glycomolecule, the method comprising: 
 providing a glycomolecule, wherein said glycomolecule has been modified by desialylation or treatment with N-Glycosidase F (PNGaseF);    adding said glycomolecule to a substrate comprising a plurality of saccharide-binding agents;    detecting glycomolecule bound to saccharide-binding agents in the plurality; and    obtaining a fingerprint for the glycomolecule based on the binding of the glycomolecule to the saccharide-binding agents.    
     
     
         2 . The method of  claim 1 , wherein said glycomolecule been modified by desialylation.  
     
     
         3 . The method of  claim 2 , wherein substantially all of the sialic acid residues have been removed from said glycomolecule.  
     
     
         4 . The method of  claim 2 , wherein the desialylation is effected by reacting said glycomolecule with a sialidase.  
     
     
         5 . The method of  claim 4 , wherein said glycomolecule is reacted with said sialidase in the presence of a protease inhibitor.  
     
     
         6 . The method of  claim 1 , wherein said glycomolecule been modified by treatment with PNGaseF.  
     
     
         7 . The method of  claim 6 , wherein substantially all of Asn-acetylglucosamine bonds have been cleaved in said glycomolecule by said PNGaseF.  
     
     
         8 . The method of  claim 1 , wherein said glycomolecule been modified by desialylation and treatment with PNGaseF.  
     
     
         9 . The method of  claim 1 , further comprising reacting said glycomolecule with a reducing agent and an alkylating agent prior to obtaining said fingerprint.  
     
     
         10 . The method of  claim 9 , wherein said glycomolecule been modified by desialylation.  
     
     
         11 . The method of  claim 10 , wherein said method comprises reacting said glycomolecule with said reducing agent and alkylating agent following desialylation.  
     
     
         12 . The method of  claim 9 , wherein said reducing agent is selected from the group consisting of β-mercaptoethanol, dithiothreitol, and mercaptethylamine.  
     
     
         13 . The method of  claim 9 , wherein said alkylating agent selected from the group consisting of iodoacetamide and iodoacetic acid.  
     
     
         14 . The method of  claim 1 , wherein all steps of said method are performed in a single container.  
     
     
         15 . The method of  claim 1 , glycomolecule is detected with a label associated with said glycomolecule.  
     
     
         16 . The method of  claim 15 , wherein said label is a fluorescent label.  
     
     
         17 . The method of  claim 16 , wherein said fluorescent label is selected from the group consisting of fluorescein isothiocyanate (FITC), rhodamine, Texas Red, and Cy5.  
     
     
         18 . The method of  claim 15 , wherein said label is added to said glycomolecule prior to adding said glycomolecule to said substrate.  
     
     
         19 . The method of  claim 15 , wherein said label is added to glycomolecule after adding said glycomolecule to said substrate.  
     
     
         20 . The method of  claim 15 , wherein said label is added to glycomolecule while adding said glycomolecule to said substrate.  
     
     
         21 . The method of  claim 15 , wherein said label is associated directly with said glycomolecule.  
     
     
         22 . The method of  claim 15 , wherein said label is associated with a second saccharide-binding agent that binds to said glycomolecule.  
     
     
         23 . The method of  claim 22 , wherein said second saccharide-binding agent is a lectin.  
     
     
         24 . The method of  claim 22 , wherein said second saccharide-binding agent is an antibody.  
     
     
         25 . The method of  claim 1 , further comprising purifying said glycomolecule prior to adding said glycomolecule to said substrate.  
     
     
         26 . The method of  claim 25 , wherein said purification is by column chromatography.  
     
     
         27 . The method of  claim 1 , wherein said glycomolecule is a glycoprotein.  
     
     
         28 . The method of  claim 27 , wherein said glycoprotein is from a cell culture medium.  
     
     
         29 . The method of  claim 27 , wherein said glycoprotein includes at least a portion of an immunoglobulin polypeptide.  
     
     
         30 . The method of  claim 29 , wherein said immunoglobulin in IgG isotype.  
     
     
         31 . The method of  claim 29 , wherein said portion comprises an Fc molecule.  
     
     
         32 . The method of  claim 1 , wherein said method comprsies treating said glycomolecule with a detergent prior to obtaining said fingerprint.  
     
     
         33 . The method of  claim 32 , wherein said detergent is an ionic detergent.  
     
     
         34 . The method of  claim 33 , wherein said detergent is sodium docecyl sulfate (SDS).  
     
     
         35 . The method of  claim 1 , wherein said substrate is a microsphere.  
     
     
         36 . The method of  claim 35 , wherein said substrate comprises a plurality of micropsheres.  
     
     
         37 . The method of  claim 35 , wherein no more than one type of saccharide-binding agent is present on said microsphere.  
     
     
         38 . The method of  claim 35 , wherein more than one type of saccharide-binding agent is present on said microsphere.  
     
     
         39 . A method for determining a glycomolecule fingerprint for a glycomolecule, the method comprising: 
 adding a glycomolecule to a substrate comprising a plurality of saccharide-binding agents;    detecting glycomolecule bound to saccharide-binding agents in the plurality; and    obtaining a fingerprint for the glycomolecule based on the binding of the glycomolecule to the saccharide-binding agents.    
     
     
         40 . The method of  claim 15 , wherein said glycomolecule is associated with a label, and bound glycomolecules are detected by identifying bound label on said substrate.  
     
     
         41 . The method of  claim 40 , label is a fluorescent label.  
     
     
         42 . The method of  claim 41 , wherein said fluorescent label is selected from the group consisting of fluorescein isothiocyanate (FITC), rhodamine, Texas Red, and Cy5.  
     
     
         43 . The method of  claim 40 , wherein said label is added to said glycomolecule prior to adding said glycomolecule to said substrate.  
     
     
         44 . The method of  claim 40 , wherein said label is added to glycomolecule after adding said glycomolecule to said substrate.  
     
     
         45 . The method of  claim 40 , wherein said label is added to glycomolecule while adding said glycomolecule to said substrate.  
     
     
         46 . The method of  claim 40 , wherein said label is associated directly with said glycomolecule.  
     
     
         47 . The method of  claim 40 , wherein said label is associated with a second saccharide-binding agent that binds to said glycomolecule.  
     
     
         48 . The method of  claim 47 , wherein said second saccharide-binding agent is a lectin.  
     
     
         49 . The method of  claim 47 , wherein said second saccharide-binding agent is an antibody.  
     
     
         50 . The method of  claim 40 , wherein said label is associated with an agent that binds to a non-carbohydrate molecule on said glycomolecule.  
     
     
         51 . The method of  claim 50 , wherein said glycomolecule is a glycoprotein and said agent binds to a peptide epitope on said glycoprotein.  
     
     
         52 . The method of  claim 49 , wherien said substrate is substantially planar.  
     
     
         53 . The method of  claim 40 , wherien said substrate is a microsphere.  
     
     
         54 . The method of  claim 53 , wherein said substrate comprises a plurality of micropsheres.  
     
     
         55 . The method of  claim 53 , wherein each microsphere comprises one type of saccharide binding agent.  
     
     
         56 . A kit for analyzing a glycomolecule, the kit comprising 
 a glycomolecule modification agent selected from the group consisting of a desialidase and a PNGase F; and    a labeling agent for labeling a glycomolecule.    
     
     
         57 . The kit of  claim 56 , further comprising a substrate comprising a plurality of saccharide-binding agents.  
     
     
         58 . The kit of  claim 56 , further comprising a reducing agent and an alkylating agent.  
     
     
         59 . The kit of  claim 56 , wherein said labeling agent binds directly to a glycomolecule.  
     
     
         60 . The kit of  claim 56 , wherein said labeling agent comprises a second saccharide-binding agent and a label that associates with said second saccharide-binding agent.  
     
     
         61 . The kit of  claim 56 , further comprising a substrate holder.  
     
     
         62 . A kit for analyzing a glycomolecule, the kit comprising 
 an antibody specifically binds to a glycoprotein; and    a labeling agent for labeling a glycomolecule.    
     
     
         63 . The kit of  claim 62 , further comprising a glycomolecule modification agent selected from the group consisting of a desialidase and a PNGase F.  
     
     
         64 . The kit of  claim 62 , further comprising a substrate comprising a plurality of saccharide-binding agents.  
     
     
         65 . The kit of  claim 62 , further comprising a reducing agent and an alkylating agent.  
     
     
         66 . The kit of  claim 62 , wherein said labeling agent binds directly to a glycomolecule.  
     
     
         67 . The kit of  claim 62 , wherein said labeling agent comprises a second saccharide-binding agent and a label that associates with said second saccharide-binding agent.  
     
     
         68 . The kit of  claim 62 , further comprising a substrate holder.  
     
     
         69 . The kit of  claim 62 , wherein said antibody binds to a polypeptide epitope on said glycoprotein.  
     
     
         70 . The kit of  claim 62 , wherein said antibody binds to a polysaccharide epitope on said glycoprotein.