Microfluidic Glycan Analysis

Abstract

Microfluidic devices and methods for analyzing glycan profiles of glycoproteins are provided. Some embodiments of the devices comprise a deglycosylation column for cleaving glycans, an optional cleaning column for removing proteins, a trapping column for enriching glycans, and a separation column for resolving glycans. The devices and methods significantly improve the speed and sensitivity of glycan analysis.

1 . A microfluidic device for removing carbohydrate from a glycoprotein, comprising:
 a deglycosylation column comprising a solid support and an enzyme immobilized to the solid support, wherein the enzyme is capable of cleaving carbohydrates from glycoproteins;   a trapping column that is capable of binding carbohydrates;   a separation column capable of separating carbohydrates; and   a plurality of inlet/outlet ports;   
       wherein said ports are configured so that when said device is coupled with a switching element that comprises at least one channel, the combination of said ports, columns and at least one channel forms a valve system that can be switched between at least a first state and a second state, the first state allowing fluid communication between the deglycosylation column and the trapping column, and the second state allowing fluid communication between the trapping column and the separation column. 
     
     
         2 . The device of  claim 1 , further comprising a cleaning column capable of binding proteins, wherein the cleaning column is configured to be connectable to the deglycosylation column and/or the trapping column by the valve system. 
     
     
         3 . The device of  claim 1 , wherein the enzyme is N-glycosidase F. 
     
     
         4 . The device of  claim 1 , wherein the solid support in the deglycosylation column comprises beads or a monolithic medium. 
     
     
         5 . The device of  claim 1 , wherein the separation column is a liquid chromatography column. 
     
     
         6 . The device of  claim 1 , wherein the separation column is a capillary electrophoresis apparatus. 
     
     
         7 . The device of  claim 1  that comprises two layers, wherein the deglycosylation column is in one layer, and the trapping column and separation column are in the other layer. 
     
     
         8 . The device of  claim 2  that comprises three layers, wherein the deglycosylation column is in a first layer, the cleaning column is in a second layer, and the trapping column and separation column are in a third layer. 
     
     
         9 . A system for analyzing a sample, comprising the device of  claim 1 , the switching element, and a mass spectrometer. 
     
     
         10 . The system of  claim 9 , wherein the mass spectrometer comprises an electrospray ion source. 
     
     
         11 . A method for analyzing the carbohydrate moieties of glycoproteins, comprising:
 applying a sample that may comprise glycoproteins to the device of  claim 1 ;   digesting the glycoproteins in the deglycosylation column to result in cleaved carbohydrates;   binding the cleaved carbohydrates to the trapping column;   eluting the cleaved carbohydrates from the trapping column; and   separating the cleaved carbohydrates with the separation column.   
     
     
         12 . The method of  claim 11 , further comprising removing proteins after the digesting with a cleaning column capable of binding proteins. 
     
     
         13 . The method of  claim 11 , wherein the cleaved carbohydrates are separated by liquid chromatography. 
     
     
         14 . The method of  claim 11 , wherein the cleaved carbohydrates are separated by capillary electrophoresis. 
     
     
         15 . The method of  claim 11 , further comprising analyzing the cleaved carbohydrates using mass spectrometry. 
     
     
         16 . The method of  claim 11 , wherein the sample contains up to 50 ng of glycoproteins. 
     
     
         17 . The method of  claim 11 , wherein the method is completed within 10 minutes. 
     
     
         18 . The method of  claim 11  that is performed under conditions that allow at least some of the cleaved carbohydrates to remain in amino glycan forms. 
     
     
         19 . The method of  claim 11 , wherein the glycoproteins are digested with N-glycosidase F. 
     
     
         20 . A kit for glycan analysis, comprising the device of  claim 1  and at least one reagent for sample dilution or column elution.