Apparatuses, Methods, Computer Program Products, And Kits for Hi-Throughput Glycan Analysis

Abstract

An apparatus for glycan analysis is disclosed. The apparatus includes a plurality of loading wells adapted to receive a plurality of samples; a plurality of capillaries arranged in correspondence with the loading wells, each of the capillaries including a first portion including a stacking gel and a second portion including a resolving gel; and a plurality of eluting wells arranged in correspondence with the capillaries and adapted to receive a portion of the samples having traversed the capillaries.

What is claimed is: 
     
         1 . An apparatus for glycan analysis, comprising:
 a plurality of loading wells adapted to receive a plurality of samples;   a plurality of capillaries arranged in correspondence with the loading wells, each of the capillaries including a first portion including a stacking gel and a second portion including a resolving gel; and   a plurality of eluting wells arranged in correspondence with the capillaries and adapted to receive a portion of the samples having traversed the capillaries.   
     
     
         2 . The apparatus of  claim 1 , wherein the stacking gel is a pre-poured stacking gel. 
     
     
         3 . The apparatus of  claim 1 , wherein the stacking gel includes between about 4% and about 8% acrylamide. 
     
     
         4 . The apparatus of  claim 1 , wherein the stacking gel includes about 6% acrylamide. 
     
     
         5 . The apparatus of  claim 1 , wherein the resolving gel is a pre-poured resolving gel. 
     
     
         6 . The apparatus of  claim 1 , wherein the resolving gel includes between about 25% acrylamide and about 35% acrylamide. 
     
     
         7 . The apparatus of  claim 1 , wherein the resolving gel includes about 30% acrylamide. 
     
     
         8 . The apparatus of  claim 1 , wherein the stacking gel includes between about 4% and about 8% acrylamide and the resolving gel includes between about 25% acrylamide and about 35% acrylamide. 
     
     
         9 . The apparatus of  claim 1 , wherein the stacking gel includes about 6% acrylamide and the resolving gel includes about 30% acrylamide. 
     
     
         10 . The apparatus of  claim 1 , wherein a total length of the first and second portions of each capillary is between about 5 cm and about 30 cm. 
     
     
         11 . The apparatus of  claim 1 , wherein a total length of the first and second portions of each capillary is about 10 cm. 
     
     
         12 . The apparatus of  claim 1 , wherein the plurality of capillaries comprise at least five capillaries arranged substantially parallel to one another, each of the capillaries including a pre-poured stacking gel arranged in the first portion of the capillary and a pre-poured resolving gel arranged in the second portion of the capillary, the capillaries further comprising first and second support structures arranged at opposite sides so as to form a single capillary array unit. 
     
     
         13 . The apparatus of  claim 1 , wherein the capillaries have an internal diameter of between about 150 micrometers and about 250 micrometers. 
     
     
         14 . The apparatus of  claim 1 , wherein the capillaries have an internal diameter of between about 0.1 millimeter and about 2.5 millimeters. 
     
     
         15 . The apparatus of  claim 1 , further comprising an ion permeable membrane arranged between the loading wells and the capillaries. 
     
     
         16 . The apparatus of  claim 1 , further comprising at least two electrodes arranged on opposite sides of the capillaries. 
     
     
         17 . The apparatus of  claim 16 , wherein the at least two electrodes are platinum electrodes. 
     
     
         18 . The apparatus of  claim 16 , wherein the at least two electrodes include a positive electrode arranged between the capillaries and the eluting wells and a negative electrode arranged between the capillaries and the loading wells. 
     
     
         19 . The apparatus of  claim 16 , further comprising a power source connected to the at least two electrodes and configured to subject at least part of the capillaries to an electric field. 
     
     
         20 . The apparatus of  claim 19 , wherein the electric field has an intensity of between about 200 V/cm and about 400 V/cm. 
     
     
         21 . The apparatus of  claim 19 , wherein the electric field has an intensity of between about 250 V/cm and about 350 V/cm. 
     
     
         22 . The apparatus of  claim 1 , further comprising a light source configured to subject the capillaries to electromagnetic radiation. 
     
     
         23 . The apparatus of  claim 22 , wherein the light source is a diode laser. 
     
     
         24 . The apparatus of  claim 22 , wherein the light source is a blue Argon ion laser. 
     
     
         25 . The apparatus of  claim 22 , wherein the light source is a yellow Krypton ion laser. 
     
     
         26 . The apparatus of  claim 22 , wherein the electromagnetic radiation is radiation having a wavelength in the range of about 400-500 nm or in the range of about 500-600 nm. 
     
     
         27 . The apparatus of  claim 22 , further comprising a fluorescence detector configured to detect fluorescence emitted from the capillaries. 
     
     
         28 . The apparatus of  claim 27 , wherein the fluorescence detector is a CCD camera. 
     
     
         29 . The apparatus of  claim 28 , further comprising a bandpass filter arranged between the capillaries and the CCD camera and configured to allow radiation having a wavelength of about 510 nm to pass. 
     
     
         30 . The apparatus of  claim 1 , wherein a largest width, depth, or height of the apparatus does not exceed about twelve inches. 
     
     
         31 . The apparatus of  claim 27 , further comprising a signal processor configured to process a signal related to fluorescence detected by the fluorescence detector. 
     
     
         32 . The apparatus of  claim 31 , wherein the signal processor is configured to generate an electrophoretogram showing peaks representing individual glycans having migrated through the capillaries so as to reveal a time point at which each glycan passed across the fluorescence detector before eluting off the end of the capillary. 
     
     
         33 . The apparatus of  claim 27 , further comprising a computer in communication with the fluorescence detector, the computer being configured to process a signal related to fluorescence detected by the fluorescence detector, wherein the computer is configured to generate an electrophoretogram showing peaks representing individual glycans having migrated through the capillaries so as to reveal a time point at which each glycan passed across the fluorescence detector before eluting off the end of the capillary. 
     
     
         34 . The apparatus of  claim 33 , wherein the computer includes or is configured to access an empirically-derived database of glycan migration times. 
     
     
         35 . The apparatus of  claim 34 , wherein the computer includes or is configured to access and run a computer program product configured to consult the empirically-derived database of glycan migration times to compare migration times obtained by running an experiment with the apparatus to identify individual glycans having migrated through the capillaries during the experiment. 
     
     
         36 . An array of capillaries for glycan analysis, comprising:
 at least five capillaries arranged substantially parallel to one another, each of the capillaries including a pre-poured stacking gel arranged in a first section of the capillary and a pre-poured resolving gel arranged in a second section of the capillary, and   first and second support structures arranged at opposite sides of the at least five capillaries such that the at least five capillaries form a single unit.   
     
     
         37 . The array of  claim 36 , wherein the stacking gel includes between about 4% and about 8% acrylamide. 
     
     
         38 . The array of  claim 36 , wherein the stacking gel includes about 6% acrylamide. 
     
     
         39 . The array of  claim 36 , wherein the resolving gel includes between about 25% acrylamide and about 35% acrylamide. 
     
     
         40 . The array of  claim 36 , wherein the resolving gel includes about 30% acrylamide. 
     
     
         41 . The array of  claim 36 , wherein the stacking gel includes between about 4% and about 8% acrylamide and the resolving gel includes between about 25% acrylamide and about 35% acrylamide. 
     
     
         42 . The array of  claim 36 , wherein the stacking gel includes about 6% acrylamide and the resolving gel includes about 30% acrylamide. 
     
     
         43 . The array of  claim 36 , wherein a length of the first section and the second section of each capillary is between about 5 cm and about 15 cm. 
     
     
         44 . The array of  claim 36 , wherein a total length of the first and second sections of each capillary is between about 10 cm and about 30 cm. 
     
     
         45 . The array of  claim 36 , wherein the capillaries have an internal diameter of between about 100 micrometers and about 300 micrometers. 
     
     
         46 . The array of  claim 36 , wherein the capillaries have an internal diameter of between about 50 micrometers and about 100 micrometers. 
     
     
         47 . The array of  claim 36 , wherein the capillaries have an internal diameter of between about 0.1 millimeter and about 2.5 millimeters. 
     
     
         48 . The array of  claim 36 , further comprising an ion permeable membrane arranged on at least one extremity of each of the capillaries. 
     
     
         49 . A library of information elements stored in a medium readable by a computer, comprising:
 a plurality of empirically-derived capillary migration times corresponding to a plurality of individual charged, fluorescently-labeled glycans having migrated through a capillary including a first portion including a stacking gel and a second portion including a resolving gel upon subjection of the capillary to an electric field; and   a migration time corresponding to a dextran ladder.   
     
     
         50 . The library of elements of  claim 49 , wherein the dextran ladder includes oligomers having an increasing number of glucose molecule, the increasing number going from one glucose molecule to about twenty glucose molecules. 
     
     
         51 . The library of elements of  claim 49 , wherein the dextran ladder includes a linear oligomer having a plurality of synthesized maltoses. 
     
     
         52 . The library of elements of  claim 49 , wherein the empirically-derived migration times corresponding to a plurality of individual glycans include empirically-derived migration times corresponding to a plurality of polysaccharides. 
     
     
         53 . The library of elements of  claim 49 , wherein the empirically-derived migration times corresponding to a plurality of individual glycans include empirically-derived migration times corresponding to a plurality of oligosaccharides. 
     
     
         54 . The library of elements of  claim 49 , wherein the empirically-derived migration times corresponding to a plurality of individual glycans include empirically-derived migration times corresponding to a plurality of proteoglycans. 
     
     
         55 . The library of elements of  claim 49 , wherein the empirically-derived migration times corresponding to a plurality of individual glycans include empirically-derived migration times corresponding to a plurality of glycoproteins. 
     
     
         56 . The library of elements of  claim 49 , wherein the empirically-derived migration times corresponding to a plurality of individual glycans include empirically-derived migration times corresponding to a plurality of glycolipids. 
     
     
         57 . The library of elements of  claim 49 , wherein the empirically-derived migration times corresponding to a plurality of individual glycans include empirically-derived migration times corresponding to a plurality of O-linked glycans. 
     
     
         58 . The library of elements of  claim 49 , wherein the empirically-derived migration times corresponding to a plurality of individual glycans include empirically-derived migration times corresponding to a plurality of N-linked glycans. 
     
     
         59 . The library of elements of  claim 49 , further comprising an empirically-derived electrophoretogram showing peaks including at least one peak corresponding to a dextran ladder. 
     
     
         60 . A method for high throughput glycan analysis, comprising:
 loading a plurality of glycoprotein samples in a plurality of loading wells;   denaturing the glycoprotein samples in the loading wells using a denaturing solution;   cleaving a glycan from each of the denatured glycoprotein samples in the loading wells using a glycan cleaving enzyme;   labeling the cleaved glycans with a charged fluorescent label;   applying an electric field configured to migrate the labeled glycans from the loading wells across an ion permeable membrane and into and along one of a plurality of capillaries arranged in correspondence with the loading wells, each of the capillaries including a first portion including a stacking gel and a second portion including a resolving gel;   exciting the labeled glycans migrating along the capillaries with a light source adapted to cause the labeled glycans to emit fluorescent radiation;   detecting fluorescent radiation emitted by the labeled glycans; and   analyzing the labeled glycans based on the detected fluorescent radiation.   
     
     
         61 . The method of  claim 60 , wherein denaturing the glycoprotein samples in the loading wells includes denaturing the glycoprotein samples using SDS. 
     
     
         62 . The method of  claim 60 , further comprising mixing each of the glycoprotein samples with a TBE buffer solution. 
     
     
         63 . The method of  claim 60 , wherein cleaving a glycan from each of the denatured glycoprotein samples includes cleaving the glycans using PNGase F. 
     
     
         64 . The method of  claim 60 , wherein cleaving a glycan from each of the denatured glycoprotein samples includes cleaving the glycans using endoglycosidase-H. 
     
     
         65 . The method of  claim 60 , wherein cleaving a glycan from each of the denatured glycoprotein samples includes cleaving the glycans using one or more of Endo D, Endo F1, Endo F2, and Endo F3. 
     
     
         66 . The method of  claim 60 , wherein cleaving a glycan from each of the denatured glycoprotein samples includes cleaving the glycans using one or more of ABS (arthrobacter ureafaciens sialidase), NAN 1 (recombinant sialidase), AMF (almond meal alpha-fucosidase), BKF (bovine kidney alpha-fucosidase), BTG (bovine testes beta-galactosidase), SPG ( streptococcus peneumoniae  beta-galactosidase), GUH ( streptococcus pneumonia  hexosaminidase, recombinant in  E. coli ), and JBM (jack bean mannosidase). 
     
     
         67 . The method of  claim 60 , wherein cleaving a glycan from each of the denatured glycoprotein samples includes cleaving the glycans using peptide-N-(N-acetyl-β-glucosaminyl)asparagine amidase. 
     
     
         68 . The method of  claim 60 , wherein cleaving the glycan using peptide-N-(N-acetyl-β-glucosaminyl)asparagine amidase includes cleaving N-linked glycans. 
     
     
         69 . The method of  claim 60 , wherein labeling the cleaved glycans with a charged fluorescent label includes labeling the cleaved glycans at a reducing end of the glycans with disodium 8-aminonaphtalene-1,3,6-trisulphonate. 
     
     
         70 . The method of  claim 60 , wherein labeling the cleaved glycans with a charged fluorescent label includes labeling the cleaved glycans at a reducing end of the glycans with potassium 7-amino-1,3-naphtalene disulfonate. 
     
     
         71 . The method of  claim 60 , wherein labeling the cleaved glycans with a charged fluorescent label includes labeling the cleaved glycans at a reducing end of the glycans with sodium 4-amino-naphtalene sulfonate. 
     
     
         72 . The method of  claim 60 , wherein labeling the cleaved glycans with a charged fluorescent label includes labeling the cleaved glycans at a reducing end of the glycans with a charged fluorescent label comprising a hydrazide functional group. 
     
     
         73 . The method of  claim 60 , wherein labeling the cleaved glycans with a charged fluorescent label includes labeling the cleaved glycans at a reducing end of the glycans with a charged fluorescent label comprising one or more of ALEXA FLUOR 350 hydrazide, ALEXA FLUOR 488 hydrazide, ALEXA FLUOR 647 hydrazide, ALEXA FLUOR 594 hydrazide, and ALEXA FLUOR 555 hydrazide. 
     
     
         74 . The method of  claim 60 , wherein labeling the cleaved glycans with a charged fluorescent label includes labeling the cleaved glycans at a reducing end of the glycans with a charged fluorescent label comprising a hydroxylamine functional group contained in one or more of ALEXA FLUOR 350 hydroxylamine, ALEXA FLUOR 488 hydroxylamine, and ALEXA FLUOR 647 hydroxylamine. 
     
     
         75 . The method of  claim 60 , wherein labeling the cleaved glycans with a charged fluorescent label includes labeling the cleaved glycans at a reducing end of the glycans with a charged fluorescent label comprising a hydrazide functional group contained in 8-hydrazide-pyene-3,6,8-trisulfonate. 
     
     
         76 . The method of  claim 60 , wherein labeling the cleaved glycans with a charged fluorescent label includes labeling the cleaved glycans at a reducing end of the glycans with a charged fluorescent label comprising a hydroxylamine functional group contained in 8-hydroxylamine-pyene-3,6,8-trisulfonate. 
     
     
         77 . The method of  claim 60 , wherein labeling the cleaved glycans with a charged fluorescent label includes labeling the cleaved glycans at a reducing end of the glycans using one or more of APTS, ANTS, ANDA, and ANSA. 
     
     
         78 . The method of  claim 60 , wherein the charged fluorescent label comprises a sulfonic acid. 
     
     
         79 . The method of  claim 60 , wherein labeling the cleaved glycans with a charged fluorescent label further includes forming a hydrazone between the hydrazide functional group and a sugar carbonyl of the glycans. 
     
     
         80 . The method of  claim 79 , wherein labeling the cleaved glycans with a charged fluorescent label further includes stabilizing the hydrazone by reduction with sodium cyanoborohydride. 
     
     
         81 . The method of  claim 60 , wherein applying an electric field includes applying an electric field having an intensity of between about 200 V/cm and about 400 V/cm. 
     
     
         82 . The method of  claim 60 , wherein applying an electric field includes applying an electric field having an intensity of between about 250 V/cm and about 350 V/cm. 
     
     
         83 . The method of  claim 60 , wherein exciting the labeled glycans includes exciting the labeled glycans with a light source. 
     
     
         84 . The method of  claim 83 , wherein the light source is a laser diode. 
     
     
         85 . The method of  claim 83 , wherein the light source has a wavelength in the range of about 400-500 nm or in the range of about 500-600 nm. 
     
     
         86 . The method of  claim 60 , wherein detecting fluorescent radiation includes detecting fluorescent radiation using a fluorescence detector. 
     
     
         87 . The method of  claim 86 , wherein detecting fluorescent radiation includes filtering fluorescent radiation directed to the fluorescence detector using a bandpass filter. 
     
     
         88 . The method of  claim 87 , wherein the band pass filter is a 510 nm bandpass filter. 
     
     
         89 . The method of  claim 86 , wherein analyzing the labeled glycans includes analyzing the labeled glycans based on an electrophoretogram generated by the fluorescence detector or by a signal processor or computer configured to process one or more signals obtained from the fluorescence detector, the electrophoretogram showing peaks representing individual glycans as they migrate along the capillaries and are detected by the fluorescence detector. 
     
     
         90 . The method of  claim 89 , wherein analyzing the labeled glycans includes comparing measured migration times to that of a fluorescently labeled dextran standard ladder and to known migration times for specific glycan structures and molecular weights previously recorded in an empirically-derived database. 
     
     
         91 . The method of  claim 90 , wherein loading the glycoprotein samples in the loading wells further includes loading a dextran standard ladder, and wherein analyzing the labeled glycans is dependent upon their migration time relative to the dextran standard ladder. 
     
     
         92 . The method of  claim 91 , wherein the dextran ladder standard includes a fluorescently labeled linear polysaccharide made of glucose molecules, including polysaccharide chains having a number of glucose molecules varying by unity increment from one glucose molecule to about twenty-three glucose molecules. 
     
     
         93 . The method of  claim 92 , wherein the dextran ladder standard is fluorescently labeled at the reducing end of the sugar chain with the charged fluorescent label comprising a hydroxylamine functional group contained in the fluorophores ALEXA FLUOR 350 hydroxylamine. 
     
     
         94 . The method of  claim 92 , wherein the dextran ladder standard is fluorescently labeled at the reducing end of the sugar chain with the charged fluorescent label comprising a hydroxylamine functional group contained in the fluorophores ALEXA FLUOR 647 hydroxylamine. 
     
     
         95 . The method of  claim 94 , further comprising, after having denatured, cleaved, and labeled the glycans, subjecting the glycans to an exoglycosidase enzyme. 
     
     
         96 . A method of making a capillary array for high throughput glycan analysis, comprising:
 providing a plurality of capillaries;   pre-pouring, into each of the capillaries, a stacking gel in a first portion and a resolving gel in a second portion; and   connecting the capillaries structurally on opposite sides such that the capillaries are arranged substantially in parallel to one another and form a single unit.   
     
     
         97 . The method of  claim 96 , wherein pre-pouring the stacking gel includes pre-pouring a stacking gel including between about 4% and about 8% acrylamide. 
     
     
         98 . The method of  claim 97 , wherein pre-pouring the stacking gel includes pre-pouring a stacking gel including about 6% acrylamide. 
     
     
         99 . The method of  claim 98 , wherein pre-pouring the resolving gel includes pre-pouring a resolving gel including between about 25% acrylamide and about 35% acrylamide. 
     
     
         100 . The method of  claim 97 , wherein pre-pouring the resolving gel includes pre-pouring a resolving gel including about 30% acrylamide. 
     
     
         101 . The method of  claim 96 , wherein a length of the first portion of each capillary is between about 5 cm and about 15 cm. 
     
     
         102 . The method of  claim 96 , wherein a length of the second portion of each capillary is between about 5 cm and about 15 cm. 
     
     
         103 . The method of  claim 96 , wherein the capillaries have an internal diameter of between about 100 micrometers and about 300 micrometers. 
     
     
         104 . The method of  claim 96 , wherein the capillaries have an internal diameter of between about 0.1 millimeter and about 2.5 millimeters. 
     
     
         105 . A method for generating a glycan database, comprising:
 empirically obtaining a plurality of empirically-derived migration times corresponding to a plurality of individual charged, fluorescently-labeled glycans having migrated through a capillary including a first portion including a stacking gel and a second portion including a resolving gel upon subjection of the capillary to an electric field; and   arranging the collected plurality of empirically-derived migration times in correspondence with a identification information of each of the plurality of individual charged, fluorescently-labeled glycans having migrated through a capillary into a database configured to be accessible by a computer.   
     
     
         106 . A method for identifying a plurality of glycans, comprising:
 labeling the glycans with a charged fluorescent label;   migrating the labeled glycans along a plurality of capillaries oriented along a substantially parallel direction into an electric field, each of the capillaries including a first portion including a stacking gel and a second portion including a resolving gel;   determining a migration time relative to a fluorescently labeled dextran standard ladder for each of the labeled glycans based on detected fluorescent radiation emitted by the labeled glycans; and   comparing the relative migration time with a database of empirically-derived migration times corresponding to a plurality of individual charged, fluorescently-labeled glycans having migrated through a capillary including a first portion including a stacking gel and a second portion including a resolving gel upon subjection of the capillary to an electric field.   
     
     
         107 . A kit for glycan analysis, comprising:
 an array of capillaries for glycan analysis, including at least five capillaries arranged substantially parallel to one another, each of the capillaries including a pre-poured stacking gel arranged in a first section of the capillary and a pre-poured resolving gel arranged in a second section of the capillary, and first and second support structures arranged at opposite sides of the at least five capillaries such that the at least five capillaries form a single unit;   a denaturing solution adapted for denaturing glycoproteins;   a glycan cleaving enzyme solution adapted for cleaving glycans; and   a fluorescent labeling solution adapted for labeling cleaved glycans.   
     
     
         108 . A kit for glycan analysis, comprising:
 a denaturing solution adapted for denaturing glycoproteins;   a glycan cleaving enzyme solution adapted for cleaving glycans; and   a fluorescent labeling solution adapted for labeling cleaved glycans.   
     
     
         109 . The kit of  claim 108 , wherein the denaturing solution includes SDS. 
     
     
         110 . The kit of  claim 108 , wherein the glycan cleaving enzyme solution includes one or more of PNGase F and endoglycosidase-H. 
     
     
         111 . The kit of  claim 108 , wherein the glycan cleaving enzyme solution includes one or more of Endo D, Endo F1, Endo F2, Endo F3, ABS (arthrobacter ureafaciens sialidase), NAN 1 (recombinant sialidase), AMF (almond meal alpha-fucosidase), BKF (bovine kidney alpha-fucosidase), BTG (bovine testes beta-galactosidase), SPG (streptococcus peneumoniae beta-galactosidase), GUH ( streptococcus pneumoniae  hexosaminidase, recombinant in  E. coli ), and JBM (jack bean mannosidase). 
     
     
         112 . The kit of  claim 108 , wherein the fluorescent labeling solution includes one or more of disodium 8-aminonaphtalene-1,3,6-trisulphonate, potassium 7-amino-1,3-naphtalene disulfonate, sodium 4-amino-naphtalene sulfonate, a charged fluorescent label comprising a hydrazide functional group, ALEXA FLUOR 350 hydrazide, ALEXA FLUOR 488 hydrazide, ALEXA FLUOR 647 hydrazide, ALEXA FLUOR 594 hydrazide, ALEXA FLUOR 555 hydrazide, ALEXA FLUOR 350 hydroxylamine, ALEXA FLUOR 488 hydroxylamine, ALEXA FLUOR. 647 hydroxylamine, 8-hydrazide-pyene-3,6,8-trisulfonate, 8-hydroxylamine-pyene-3,6,8-trisulfonate, APTS, ANTS, ANDA, and ANSA. 
     
     
         113 . The method of  claim 60 , wherein one or more of the plurality of glycoprotein samples are recombinant proteins. 
     
     
         114 . The method of  claim 113 , wherein one or more of the plurality of glycoprotein samples are antibodies. 
     
     
         115 . The method of  claim 113 , wherein one or more of the plurality of glycoprotein samples are intracellular proteins, membrane associated proteins or secreted proteins. 
     
     
         116 . The method of  claim 114 , wherein one or more of the glycoprotein samples are analyzed for reduced core fucosylation 
     
     
         117 . The method of  claim 116 , wherein an antibody-producing cell is grown in a culture medium treated with an agent that alters core fucosylation. 
     
     
         118 . The method of  claim 117 , wherein the core fucosylation levels of one or more of the glycoprotein samples are compared before and after treatment. 
     
     
         119 . The apparatus of  claim 1 , which identifies a core fucosylated glycoprotein from a non-fucosylated glycoprotein. 
     
     
         120 . The apparatus of  claim 1 , which differentiates between a 5% to 0.1% fucosylated glycoprotein. 
     
     
         121 . The method of  claim 60 , wherein labeling the cleaved glycans with a charged fluorescent label further includes forming a oxime between the hydroxylamine functional group and a sugar carbonyl of the glycans.