Glycosylation Methods and Analysis Sigma

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GlycoproteinAnalysis

GlycanClassification

GlycoproteinPurification

DeglycosylationStrategies

Detection

Glycan Analysis

Product Directory

M A N U A LAuthors:Robert GatesElner RathboneLisa MastersonIan WrightAsgar Electricwala

1st Edition

Glycoprotein Analysis

Application NotePNGase F Enzyme: Efficient N-Deglycosylation in Glycoprotein AnalysisBy Elner Rathbone, Asgar Electricwala, and Ian WrightSigma-Aldrich Corporation, Poole, England

IntroductionGlycosylation is one of the most common post-translationalmodifications of proteins in eukaryotic cells. These glyco-proteins are involved in a wide range of biological func-tions such as receptor binding, cell signaling, immunerecognition, inflammation, and pathogenicity. Mammalianglycoproteins contain three major types of oligosaccha-rides (glycans): N-linked, O-linked, and glycosylphosphatidyli-nositol (GPI) lipid anchors. N-Linked glycans are attachedto the protein backbone via an amide bond to an asparagineresidue in an Asn-Xaa-Ser/Thr motif, where X can be anyamino acid, except Pro. O-Linked glycans are linked via thehydroxyl group of serine or threonine. Variation in thedegrees of saturation at available glycosylation sites resultsin heterogeneity in the mass and charge of glycoproteins.

To study the structure and function of a glycoprotein, it isoften desirable to remove all or just a select class of glycans.This approach allows the assignment of specific biologicalfunctions to particular components of the glycoprotein.The removal of N-linked glycans from glycoproteins elim-inates heterogeneity in matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS analysis. Also,removal of glycans may enhance or reduce the bloodclearance rate and/or potency of a glycoprotein thera-peutic. Although sites of potential N-glycosylation can bepredicted from the consensus sequence Asn-Xaa-Ser/Thr,it cannot be assumed that a site will actually be glycosy-lated. Therefore the sites of glycan attachment must beidentified and characterized by analytical procedures.

Peptide-N-glycosidase F (PNGase F) is one of the most widelyused enzymes for the deglycosylation of glycoproteins.The enzyme releases asparagine-linked (N-linked) oligosac-charides from glycoproteins and glycopeptides. A tripeptidewith the glycan-linked asparagine as the central residueis the minimum substrate for PNGase F. The glycan canbe a high-mannose, hybrid, or complex type. However, N-glycans with fucose linked a(1,3) to the Asn-bound N-acetylglucosamine are resistant to the action of PNGase F.

MALDI-TOF MS is a widely used technique for rapid iden-tification of proteins separated by gel electrophoresis.Glycopeptides, however, suffer from signal suppression

during MS analysis due to the heterogeneity of theattached glycans. In this application note, we have usedPNGase F enzyme (Product Code P 7367) for the deglyco-sylation of a model glycoprotein, a1-antitrypsin, and haveidentified and localized N-glycosylation sites by MALDI-MSpeptide mapping. The GlycoProfile I Enzymatic In-Gel N-Deglycosylation Kit (Product Code PP 0200) convenientlyprovides quality-tested reagents and a detailed protocolfor in-gel protein deglycosylation.

Efficient and convenient in-gel protein deglycosylationA specific example demonstrating the use of the enzymeis the deglycosylation of the glycoprotein a1-antitrypsin.After reduction and alkylation (using tributylphosphine andiodoacetamide contained in the ProteoPrep Reductionand Alkylation Kit, Product Code PROT-RA), samples aremixed with SDS-PAGE sample buffer and separated on10% BisTris NuPAGE gel (Invitrogen Corp., Carlsbad, CA)using MOPS running buffer. The gel is stained with 0.1%Coomassie blue stain and destained.

The glycoprotein band from the 1D gel (or a spot from a 2D gel) is carefully cut out and sliced into sections. Thepieces are destained, dried, and incubated with PNGase F.The liquid surrounding the gel pieces is removed andeither discarded or retained for gylcan analysis if required.The dried gel pieces are incubated with trypsin (ProductCode T 6567) and the surrounding liquid containing thetryptic peptides is removed for further analysis.

Facile sample preparation for mass spectrometrySample preparation is simplified since the enzyme formu-lation does not contain detergents or stabilizers thatcould interfere with analysis by MS. The solution of trypticpeptides is concentrated and desalted on a C18 ZipTip

(Millipore Corporation, Billerica, MA) prior to analysis.MS analysis was carried out on a MALDI-TOF instrumentfitted with a 337-nm UV laser. Peptides were analyzed in positive ion reflectron mode using a-cyano-4-hydroxy-cinnaminic acid as the matrix. Spectra were acquired over a mass range of 4000 m/z with matrix suppressionset at 800 mass units. Data analysis was carried out usingProteinLynx software (Waters Corporation, Milford, MA).

SummaryPNGase F is an effective enzyme for the release of N-linkedglycans from glycoproteins, in gel or in solution. The for-mulation employed for PNGase F (Product Code P 7367)provides a convenient, stable, freeze-dried product thatis compatible with MS analysis. This enzyme is also includedin the GlycoProfile In-Gel and GlycoProfile In-SolutionDeglycosylation Kits, which are composed of Proteomicsgrade, high purity components for N-deglycosylation andtryptic digestion.

Overview

Key to Monosaccharide Symbols, Abbreviations and Projections . . . . . . . . . . . . 02

Classification and Structure of Glycan Components

N-Linked Glycans . . . . . . . . . . . . . . . . . . . 05O-Linked Glycans . . . . . . . . . . . . . . . . . . . 09GPI Anchored Proteins . . . . . . . . . . . . . . . 12

Glycoprotein Purificationm-Aminophenylboronate Matrices . . . . . . . . 13Lectin Matrices . . . . . . . . . . . . . . . . . . . . . . . . 13

Glycoprotein DetectionColorimetric Detection on PAGE and Blots . . . 15Fluorescent Detection on PAGE . . . . . . . . . . . 16Biotin Labeling on Blots . . . . . . . . . . . . . . . . . 17Glycoprotein Standards . . . . . . . . . . . . . . . . . 18

Chemical Deglycosylation StrategiesHydrazinolysis. . . . . . . . . . . . . . . . . . . . . . . . . 22Alkaline b-Elimination . . . . . . . . . . . . . . . . . . 23Trifluoromethanesulfonic Acid. . . . . . . . . . . . 23

Enzymatic Deglycosylation StrategiesN-Linked Glycan Enzymatic Hydrolysis . . . . . . 24Native and Sequential Hydrolysis . . . . . . . . . . 25O-Linked Glycan Enzymatic Hydrolysis . . . . . . 29Endoglycosidases . . . . . . . . . . . . . . . . . . . . . . 32

Glycan Labeling and AnalysisGlycan Labeling . . . . . . . . . . . . . . . . . . . . . . . 36HPLC Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 37Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . 39NMR Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 45Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . 45

Glycan and Carbohydrate StandardsN-Linked High Mannose. . . . . . . . . . . . . . . . . 47N-Linked Hybrid . . . . . . . . . . . . . . . . . . . . . . . 49N-Linked Complex . . . . . . . . . . . . . . . . . . . . . 49N-Linked Fragments . . . . . . . . . . . . . . . . . . . . 55O-Linked Neutral Glycans . . . . . . . . . . . . . . . . 57O-Linked Sialylated Glycans . . . . . . . . . . . . . . 60Blood Group Antigens . . . . . . . . . . . . . . . . . . 63Lewis and Cell Adhesion Glycans . . . . . . . . . . 65Gal a(1,3) Gal Antigens . . . . . . . . . . . . . . . . . 67Monosaccharides . . . . . . . . . . . . . . . . . . . . . . 67

Glycobiology Product DirectoryAdditional Information Sources: Enzyme Explorer. . . . . . . . . . . . . . . . . . . . . . . 68Complete List of Glycolytic Enzymes . . . . . . . 69GPI Anchor Enzymes. . . . . . . . . . . . . . . . . . . . 74Glycosyltransferases . . . . . . . . . . . . . . . . . . . . 74Inhibitors of Carbohydrate Metabolism . . . . . . 75Glycolytic Enzyme Substrates . . . . . . . . . . . . . 76Neoglycoproteins . . . . . . . . . . . . . . . . . . . . . . 79Lectins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Table of Contents

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Key to Monosaccharide Symbols, Abbreviations, and Projections

Symbols, Structure Projections, and Abbreviations for Monosaccharides

Monosaccharide 3-D Chair projection Haworth projection Fischer projection Symbol

b-D-Glucose (Glc)

b-D-Mannose (Man)

O OH

CH2OH

OHOH

OH

CH2OH

HHO

OHH

HHO

OHH

O

H

GlcO

HO

HO OHOH

OH

O OH

CH2OH

OHOH OH

CH2OH

HHO

HHO

HHO

OHH

O

H

OHO

HO

HO

OH

OH

Man

One of the distinguishing features of theproteome in eukaryotic cells is that mostproteins are subject to post-translationalmodification, of which glycosylation is themost common form. It is estimated thatmore than half of all proteins that havebeen characterized are glycoproteins. Thecarbohydrate components of glycoproteinsperform critical biological functions inprotein sorting, immune and receptorrecognition, inflammation, pathogenicity,metastasis, and other cellular processes.

Mammalian glycoproteins contain threemajor types of oligosaccharides (glycans):N-linked, O-linked, and glycosylphosphatidyl-inositol (GPI) lipid anchors. N-Linked glycansare linked to the protein backbone via anamide bond to asparagine residues in anAsn-X-Ser/Thr motif, where X can be anyamino acid, except Pro. O-Linked glycansare linked to the hydroxyl group of serine

or threonine. GPI-anchored proteins areattached at their carboxy-terminus through aphosphodiester linkage of phosphoethanol-amine to a trimannosyl glucosamine corestructure. The reducing end of the lattermoiety is bound to the hydrophobic regionof the membrane via a phosphatidyl-inositol group.

Variations in structure and degree of gly-cosylation site saturation can contribute tooverall mass heterogeneity. The terminalresidues of these glycans are commonly N-acetylneuraminic acid (sialic acids). Thedegree of sialylation affects both the mass and charge of a glycoprotein. Other modi-fications to the protein such as sulfation or phosphorylation also affect charge. O-Linked glycans often have lower massthan N-linked structures, but can be moreabundant and heterogeneous.

Overview

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b-D-Galactose (Gal)

b-D-N-Acetylglucosamine (GlcNAc)

b-D-N-Acetylgalactosamine (GalNAc)

b-D-Xylose (Xyl)

O OH

CH2OH

OH

OH

NHAc

O OH

OHOH

OH

Xyl

O OH

CH2OH

OHOH

NHAc

CH2OH

HHO

NHAcH

HHO

OHH

O

H

H

HHO

OHH

HHO

OHH

O

H

GalNAc

CH2OH

HHO

NHAcH

HHO

HHO

O

H

GlcNAc

OHO

HO NHAcOH

OH

OHO

HO OHOH

OHO

HO NHAcOH

OH

O OH

CH2OH

OH

OH

OHCH2OH

HHO

OHH

HHO

HHO

O

H

OHO

HO OHOH

OH

Gal

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a-N-Acetylneuraminic acid Sialic Acid (NeuNAc)

b-D-Glucuronic acid (GlcA)

a-L-Iduronic acid (IdoA)

a-L-Fucose (Fuc)

CO2H

HHO

OHH

HHO

OHH

O

H

IdoA

GlcA

CH2OH

O

H

HO CO2H

H H

H OH

H OH

H OH

AcHN H

OHO

HO OHOH

CO2H O OH

CH2OH

OHOH

OH

Fuc

CO2H

HO H

H OH

HO H

H OH

O

H

OHO

HOHO2C

OHOH

O

OHOHCH2OH

OHOH

O CO2H

OH

CH2OH

AcHN

OH

OHOHOHCO2H

OHHO

HOOH

AcHN HO O

CH3

HO H

HO H

H OH

H OH

O

H

NeuNAc

O

OH

CH3OH

OH

OHH3C

HOHO

OH

OHO

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Basic N-linked Structure

Classification and Structure of Glycan ComponentsN-Linked GlycansAll eukaryotic cells express N-linked glycoproteins. Proteinglycosylation of N-linked glycans is actually a co-translationalevent, occurring during protein synthesis. N-linked gly-cosylation requires the consensus sequence Asn-X-Ser/Thr.Glycosylation occurs most often when this consensussequence occurs in a loop in the peptide. Oligosaccharideintermediates destined for protein incorporation are syn-thesized by a series of transferases on the cytoplasmicside of the endoplasmic recticulum (ER) while linked tothe dolichol lipid. Following the addition of a specificnumber of mannose and glucose molecules, the orienta-tion of the dolichol precursor and its attached glycan shiftsto the lumen of the ER where further enzymatic modifi-cation occurs. The completed oligosaccharide is thentransferred from the dolichol precursor to the Asn of thetarget glycoprotein by oligosaccharyltransferase (OST).Further processing includes trimming of residues such asglucose and mannose, and addition of new residues viatransferases in the ER and, to a great extent, in the Golgi.In the Golgi, high mannose N-glycans can be convertedto a variety of complex and hydrid forms which are uniqueto vertibrates.

Inhibition or elimination of glycosylation in the study ofN-linked glycans can be brought about by a number ofcompounds. In the presence of compactin, coenzyme Q,and exogenous cholesterol, N-glycosylation is greatlyinhibited. Treatment with tunicamycin completely blocksdeglycosylation in that it inhibits GlcNAc C-1-phosphotrans-ferase, which is critical in the formation of the dolicholprecursor necessary for synthesizing of N-glycans.

The diverse assortment of N-linked glycans are based onthe common core pentasaccharide, Man3GlcNAc2. Furtherprocessing in the Golgi results in three main classes of N-linked glycan sub-types; High-mannose, Hybrid, andComplex. Complex glycans contain the common triman-nosyl core. Additional monosaccharides may occur inrepeating lactosamine units. Additional modifications mayinclude a bisecting GlcNAc at the mannosyl core and/or a fucosyl residue on the innermost GlcNAc. Complexglycans exist in bi-, tri- and tetraantennary forms.

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High-Mannose Structure

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Hybrid Structure

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Complex Structure (tetraantennary)

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O-Linked GlycansO-Linked glycans are usually attached to the peptide chainthrough serine or threonine residues. O-Linked glycosyla-tion is a true post-translational event and does not requirea consensus sequence and no oligosaccharide precursor is required for protein transfer. The most common typeof O-linked glycans contain an initial GalNAc residue (orTn epitope), these are commonly referred to as mucin-typeglycans. Other O-linked glycans include glucosamine, xylose,galactose, fucose, or manose as the initial sugar bound tothe Ser/Thr residues. O-Linked glycoproteins are usuallylarge proteins (>200 kDa) that are commonly bianttennarywith comparatively less branching than N-glycans.Glycosylation generally occurs in high-density clustersand may contribute as much as 50-80% to the overall mass.O-Linked glycans tend to be very heterogeneous, hencethey are generally classified by their core structure. Non-elongated O-GlcNAc groups have been recently shown tobe related to phosphorylation states and dynamic processing

related to cell signaling events in the cell. O-Linked glycansare prevalent in most secretory cells and tissues. They are present in high concentrations in the zona pelucidasurrounding mammalian eggs and may funtion as spermreceptors (ZP3 glycoprotein). O-Linked glycans are alsoinvolved in hematopoiesis, inflammation response mech-anisms, and the formation of ABO blood antigens.

Elongation and termination of O-linked glycans is carriedout by several glycosyltransferases. One notable corestructure is the Gal-b(1-3)GalNAc (core 1) sequence thathas antigenic properties. Termination of O-linked glycansusually includes Gal, GlcNAc, GalNAc, Fuc, or sialic acid.By far the most common modification of the core Gal-b(1-3)-GalNAc is mono-, di-, or trisialylation. A lesscommon, but widely distributed O-linked hexasaccharidestructure contains b(1-4)-linked Gal and b(1-6)-linkedGlcNAc as well as sialic acid.

Di- and Trisialated O-Linked Core

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O-Linked Core 2 Hexasaccharide

Core 1

Core 2

Core 3

Core 4

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Core 5

Core 6

Core 7

Core 8

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GPI Anchor GlycoproteinsGlycosylphosphatidylinisotol (GPI) anchored proteins aremembrane bound proteins found throughout the animalkingdom. GPI anchored proteins are linked at their carboxy-terminus through a phosphodiester linkage of phospho-ethanolamine to a trimannosyl-non-acetylated glucosamine(Man3-GlcN) core. The reducing end of GlcN is linked tophosphatidylinositol (PI). PI is then anchored throughanother phosphodiester linkage to the cell membranethrough its hydrophobic region. Intermediate forms arealso present in high concentrations in microsomal prepa-rations. The Man3-GlcN oligosaccharide core may undergovarious modifications during secretion from the cell.

Their functionality ranges from enzymatic to antigenicand adhesion. They contribute to the overall organizationof membrane bound proteins and are important in apicalprotein postioning. GPI-anchored proteins also play acritical role in a variety of receptor-mediated signaltransduction pathways.

Release of GPI anchored proteins can be accomplished bytreatment with Phospholipase C, Phosphatidylinositol-specific (PLC-PI) (Product Codes P 5542 and P 8804). Theenzyme specifically hydrolyzes the phosphodiester bond of phosphatidylinositol to form a free 1,2-diacylglyceroland glycopeptide-bound inositol cyclic-1,2-phosphate.

GPI Anchor

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Lectin Matrices(For additional Lectins see page 80)

Concanavalin A matrices bind specifically to mannosyland glucosyl residues of polysaccharides and glyco-proteins. Unmodified hydroxyl groups at the C3, C4, andC6 positions of D-glucopyranosyl or D-mannopyranosylrings may be essential for binding. Con A matrices havebeen used with SDS (0.05%) and TRITON X-100.

Procedure Equilibration and Binding: Pre-wash column with 5 column volumes of wash

solution (1 M NaCl, 5 mM MgCl2, 5 mM MnCl2, and 5 mM CaCl2).

Equilibrate column in the buffer of choice (pH rangesgenerally between 6.5 to 7.5 although buffers as low aspH 4.1 and as high as 9.0 have been used successfully).A commonly used starting buffer is 20 mM Tris, pH 7.4,containing 0.5 M NaCl.

Load sample solution in equilibration buffer (proteinconcentrations 1-20 mg/ml, free of particulates).

Wash the resin with equilibration buffer until eluent isprotein free.

Elution: Elute the target protein with gradient or step-wise

elution with methyl a-D-glucopyranoside or methyl a-D-mannopyranoside, glucose, or mannose (5 mM -500 mM).

Maximum recovery and cleaning of the resin may beachieved by using 1 M sucrose, glucose, mannose, orcorresponding a-methyl glycoside. The addition ofchaotropic agents (0.5 M to 6 M) may also be requiredfor maximum recovery, but these denaturing conditionsmay severely damage the resin. Therefore they shouldonly be used as a last resort.

Glycoprotein Purification

Purification of proteins selectively utilizing their glycancomponent as a capture target is commonly done utiliz-ing affinity chromatography. The most popular affinitymatrices are m-aminophenylboronic acid agarose and the immobilized lectins, Con A and Wheat Germ.

m-Aminophenylboronic Acid Matricesm-Aminophenylboronic acid matrices are capable offorming temporary bonds with any molecule thatcontains a 1,2-cis-diol group.

Procedure Equilibration buffers should be of low ionic strength,

with pH 7-9. For a column volume of 1 ml, apply 1-2 mg of protein in

approximately 250 ml of buffer: 50 mM taurine/NaOH,pH 8.7, containing 20 mM MgCl2.

Optimize the column flow rate to 2 ml/hour, collecting 2 ml fractions.

Elute the bound protein using the same buffer with 50 mM sorbitol or 50 mM Tris/HCl added.

References 1. Mallia, A.K. et al., Anal. Letters, 14 (B8), 649-661 (1981).2. Immobilized Affinity Ligand Techniques, Hermanson, G.T., et al.,

Eds. (Academic Press, 1992), pp. 338, 339-392.3. Affinity Chromatography: A Practical Approach, P.D.G Dean, W.S.

Johnson and F. A. Middle, Eds., (IRL Press, 1985), p. 133.

Aminophenylboronate Matrices

A 8530 m-Aminophenylboronic Acid Matrix: cross-linked 6% beaded agaroseAffinity Medium Activation: epoxy, with attachment through the amino group, with a 12-atom spacer

Ligand immobilized: 5-20 mmoles per mlForm: (light pink) suspension in 0.5 M NaCl, with 0.1 M sodium acetate, pH 5.0 Synonym: PBA-agarose

A 4046 m-Aminophenylboronic Acid Matrix: acrylic beadsAffinity Medium Activation: oxirane, with attachment through the amino group, with a 5-atom spacer

Ligand immobilized: 300-600 mmoles per gramForm: lyophilized powder Swelling: 1 g swells to approximately 4 ml

A 8312 m-Aminophenylboronic Acid Matrix: 6% beaded agaroseAffinity Medium Activation: epichlorohydrin, with attachment through amino group with a 9-atom spacer

Ligand immobilized: 40-90 mmoles per mlBinding Capacity: 8-14 mg Peroxidase Type VI per mlForm: suspension in water containing 0.002% chlorhexidine diacetate.

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References1. Yahara, I. and Edelman, G.M., Restriction of the mobility of lym-

phocyte immunoglobulin receptors by concanavalin A. Proc. Nat.Acad. Sci. USA, 69(3), 608-612 (1972).

2. Li, Y., et al., The p185neu-containing Glycoprotein Complex of aMicrofilament-associated Signal Transduction Particle. J. Biol.Chem., 274(36), 25651-25658 (1999).

3. Romero, P.A., et al., Glycoprotein biosynthesis in Saccharomycescerevisiae. Partial purification of the alpha-1,6-mannosyltrans-ferase that initiates outer chain synthesis. Glycobiology, 4(2), 135-140 (1994).

Glycoprotein Purification

Wheat Germ (Triticum vulgaris) matrices are specific forGlcNAc2 or NeuNAc residues.

Procedure Equilibration and Binding: Wash and equillibrate column with 5 column volumes

of wash solution (0.05 M sodium phosphate, pH 7.0,containing 0.2 M NaCl).

Load sample solution in equilibration buffer (proteinconcentrations 1-20 mg/ml, free of particulates).

Wash the resin with equilibration buffer until eluent isprotein free.

Elution: Elute the target protein with gradient or step-wise

elution with equilibration buffer containing 100 mg/mlN-acetylglucosamine (Product Code A 8625).

Con A Matrices

C 9017 Concanavalin A Immobilized Matrix: Sepharose 4BActivation: cyanogen bromideLigand immobilized: 10-15 mg per mlBinding Capacity: 20-45 mg thyroglobulin per mlForm: suspension in 0.1 M acetate buffer, pH 6.0, containing 1 M NaCl, 1 mM each ofCaCl2, MgCl2, and MnCl2 and 0.01% thimerosal

C 6170 Concanavalin A Immobilized Matrix: 4% beaded agaroseActivation: cyanogen bromideLigand immobilized: approx. 15 mg per mlBinding Capacity: 6 mg yeast mannan per mlForm: suspension in 0.1 M acetate buffer, pH 6.0, containing 1 M NaCl, 1 mM each ofCaCl2, MgCl2, and MnCl2 and 0.02% thimerosal

WGA Matrices

L 1882 Wheat Germ (Triticum vulgaris) Matrix: Cross-Linked 4% beaded agaroseLectin Immobilized Activation: cyanogen bromide

Ligand immobilized: 5-10 mg per mlForm: Suspension in 1.0 M NaCl and 0.02% thimerosal

L 1394 Wheat Germ (Triticum vulgaris) Matrix: 6% agarose macrobeadsLectin Immobilized Activation: cyanogen bromide

Ligand immobilized: Approx. 6 mg per mlBinding Capacity: 1-2 mg ovomucoid per mlForm: Suspension in 0.9% NaCl and 0.01% thimerosal

References1. Lotan, R., et al., Activities of lectins and their immobilized deriv-

atives in detergent solutions. Implications on the use of lectinaffinity chromatography for the purification of membrane glyco-proteins. Biochem., 16, 1787-1794 (1977).

2. Janicott, M., et al., The insulin-like growth factor 1 (IGF-1) recep-tor is responsible for mediating the effects of insulin, IGF-1, andIGF-2 in Xenopus laevis oocytes. J. Biol. Chem., 266, 9382 (1991).

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Glycoprotein Detection

Initial detection of glycoproteins in vitro is routinely accomplished on SDS-PAGE gels andWestern blots. Cellular localization of glycoproteins is normally accomplished utilizinglectin fluorescent conjugates.

For a complete list of lectins including fluorescent labeled lectins, see page 80.

Colorimetric Detection on PAGE andWestern blotsGlycoprotein Detection KitProduct Code GLYCO-PRO

Product DescriptionThe Glycoprotein Detection Kit provides a system to easilydetect the sugar moieties of glycoproteins on SDS-PAGE oron Western blotting membranes. This detection system isa modification of Periodic acid-Schiff (PAS) methods andyields magenta bands with a light pink or colorless back-ground. The detection limits have been found to be 25-100 ng of carbohydrates depending on the natureand the degree of glycosylation of proteins. Peroxidase

from horseradish, reported as having a carbohydratecontent of approximately 16%, is used as a positivecontrol in the kit. The table below describes the stepsand time required when utilizing this kit.

Contains sufficient materials to stain 10 mini gels (8 x 10 cm) or 5 large gels (16 x 18 cm) or same sizes of blotting membranes.

Components

Oxidation Component (Periodic Acid)

Reduction Component (Sodium Metabisulfite)

Schiffs Reagent, Fuchsin-Sulfite Reagent

Peroxidase

Steps Time for gel thickness 0.5-0.75 mm or for membrane Time for gel thickness 1.0-1.5 mm

1. Fixing 30 min. 60 min.

2. Washing 2 x 10 min. 2 x 20 min.

3. Oxidation 30 min. 60 min.

4. Washing 2 x 10 min. 2 x 20 min.

5. Staining 1-2 hours or until bands turn magenta 1-2 hours or until bands turn magenta

6. Reduction 60 min. 120 min.

7. Washing Band color will intensify with changes of fresh water Band color will intensify with changes of fresh water

8. Storage overnight overnight

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Fluorescent Detection on PAGE GelsGlycoProfile IIIFluorescent Glycoprotein Detection Kit 8 Product Code PP0300

Product DescriptionGlycoProfile III is designed for the fluorescent in-geldetection of glycosylated proteins utilizing standard UV-transillumination. Following SDS-PAGE, proteins arefixed in the gel with an acetic acid/methanol solution.The carbohydrates on the proteins are oxidized to alde-hydes with periodic acid. A hydrazide dye is reacted withthe aldehydes, forming a stable fluorescent conjugate.This allows for the specific, sensitive detection of theglycoproteins directly in gels. This kit can also be used to detect glycoproteins after Western transfer to PVDFmembranes.

The classical method for in-gel carbohydrate detectionuses Periodic Acid-Schiff reagent (PAS). It has a detectionlimit of 25-100 ng of carbohydrate. PAS staining of glyco-proteins is very selective, but lacks the sensitivity of fluo-rescent detection (5-25 ng of carbohydrate). The limit ofdetection varies with the glycoprotein and the degreeand type of glycosylation. Typical detection limits observedwith the ProteoProfile PTM Marker (P 1745) are 150 ngof ovalbumin (5 ng of carbohydrate) and 150 ng of RNase B(30 ng carbohydrate). The detection limits are 5-10 timeslower than those observed with the Periodic Acid-Schiffreagent.

Sufficient reagents are supplied for 10 mini gels.

Components

Oxidation Reagent

Glycoprotein Staining Reagent

Staining Buffer

ProteoProfile PTM Marker

Designed as both a positive and negative control for SDS-PAGE gelsand Western blots of proteins with post-translation modifications.

SpecificityAlthough this staining procedure is quite selective forglycoproteins, some non-specific protein staining mayoccur and may be more pronounced in some gel formu-lations. Staining the gel with EZBlue Gel StainingReagent after fluorescent imaging will allow identifi-cation of non-specifically stained proteins.

An alternative method is to run duplicate gels and fluo-rescently stain the second gel omitting the oxidationstep. Any fluorescent staining will be non-specific.

Figure 1. PTM Marker (2 ml of a 6-fold dilution), containing glycosy-lated and non-glycosylated proteins, was separated by electrophoresison a 420% SDS-PAGE gel. The gel was stained for glycoproteinswith GlycoProfile III (left), imaged, and then stained for total proteinwith EZBlue Gel Staining Reagent (right). The glycoproteins appearas bright fluorescent bands. Each band represents approximately 300 ng of protein.

Figure 2. Mouse IgG and rabbit IgG were separated on a 420%SDS-PAGE gel and stained in the same manner as the gel in Figure 1.The IgG heavy chains, which are glycosylated, react strongly with the fluorescent detection reagent. 2.5 mg of protein was applied to each lane.

IgG Heavy Chain (glycosylated)

IgG Light Chain

Mo

use

Rab

bit

Mo

use

Rab

bit

BSA (not glycosylated, not phosphorylated)

Ovalbumin (glycosylated and phosphorylated)

b-Casein (not glycosylated, phosphorylated)

RNase B (glycosylated, not phosphorylated)

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Detection of Biotin LabeledGlycoproteins on Western blotsBiotin-hydrazide modification of periodate oxidized gly-cans can be utilized to label glycoproteins on Westernblots. The blots can then be probed with streptavidin-peroxidase.1 Detection can be accomplished with either

colorimetric TMB or chemiluminescent CPS-1 peroxidasesubstrate solutions. Alkaline phosphatase conjugatedstreptavidin can also be used to probe the blots. Typicaldetection limits for glycoproteins using this is methodare approximately 50-100 ng.

Optimal results are obtained on PDF membranes.

Detection Products

B 7639 Biotin Hydrazide

S 5512 Streptavidin, Peroxidase Labeled

S 1878 Sodium (meta) Periodate

T 0565 Tetramethylbenzidine Liquid Substrate System

CPS1-60 Chemiluminescent Peroxidase Substrate 60 ml



Reference1. Bayer, E.A., et al., Meth. Enzymol., 184, 415 (1990).

O

OHOH

O

OO

O

CH CH

N N

NH NH

O = C C = O

H2C H2C

H2C H2C

CH2 CH2

CH2 CH2

Periodate Biotin Hydrazide

Glycan PeriodateOxidizedGlycan

Biotin Labeled Glycan

NH2CH2 CH2 CH2 CH2 C NH

O= = = = = =

=

H H

O

S

HN

NH

HN

NHH

H

H

H

O OS S

HN NH

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Preferred Glycoprotein Standards

I 0408 Invertase Glycoprotein Standard Invertase is an enzyme that catalyzes the hydrolysis of sucrose into fructose and glucose. 8 Invertase Glycoprotein Standard is the periplasmic (glycosylated form, external invertase)

with 50% of its mass as polymannan. The periplasmic invertase molecule can exist in anumber of association states each a multiple of the core glycosylated monomer, a 60 kDapeptide plus oligosaccharide chains and, depending on extraction, purification, and storageconditions, will exist as a dimer, tetramer, hexamer, or octamer. Since yeast can provide analternative system for protein glycosylation that is similar to mammalian systems, periplas-mic invertase is often used as a model for the study of the function of oligosaccharides inglycoproteins and for studies on glycoprotein biosynthesis.

R 1153 RNase B Glycoprotein Standard Bovine pancreatic Ribonuclease B (RNase B) is a glycoprotein that contains only N-linked 8 glycans. It is a globular protein composed of a single domain that occurs naturally as a

lesser component in a mixture along with Ribonuclease A (RNase A) which is the non-glycosylated core form. RNase B contains a single glycosylation site at Asn34 at which from five to nine mannose residues are attached to the chitobiose core, i.e. Man5GlcNAc2,Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2 and Man9GlcNAc2. Due to the heterogeneity in the glycosylation at Asn34, RNase B exists as five glycosylated variants, with a molecularweight of approximately 15,000 Da.

Key Glycoproteins for Scientific Research

G 9885 a1-Acid Glycoprotein, human 99%, Purified from Cohn Fraction VI

A 5566 Monoclonal Anti-a1-Acid produced in mouse, Clone AGP-47, Ascites fluid, liquidGlycoprotein antibody

A 0534 Anti-a1-Acid Glycoprotein antibody produced in rabbit, IgG fraction of antiserum, Lyophilized powder

A 3418 Anti-a1-Acid Glycoprotein antibody produced in goat, Whole antiserum, liquid

A 9285 a1-Antichymotrypsin approx. 95% (SDS-PAGE) Lyophilized powderfrom human plasma Glycoprotein is specific inhibitor of chymotrypsin-like serine proteases.

Contains Tris buffer salt and NaCl

A 9024 a1-Antitrypsin Salt-free, lyophilized powder, a1-Proteinase inhibitor, serine protease inhibitor; inhibits trypsin, from human plasma chymotrypsin, pancreatic and granulocytic elastase, and acrosin. Effective concentration

equimolar with proteinase. Chromatographically prepared and partially purified.

G 0516 a2-hs-Glycoprotein minimum 90% (SDS-PAGE) Lyophilized from 20 mM Tris-HCl, pH 8.0, with 200 mM NaClfrom human plasma

A 1960 Aggrecan Lyophilized powder, salt essentially freefrom bovine articular cartilage sterile-filtered (dialyzed against water)

A 2512 Albumin Grade VI approx. 99% (agarose gel electrophoresis) Lyophilized powderfrom chicken egg white Extent of labeling 5-6 mol mannose per mol ovalbumin protein(Ovalbumin)

A 5503 Albumin Grade V minimum 98% (agarose gel electrophoresis) Lyophilized powderfrom chicken egg white salt essentially free(Ovalbumin)

A 5378 Albumin Grade III minimum 90% (agarose gel electrophoresis) Lyophilized powderfrom chicken egg white (Ovalbumin)

A 4781 Asialofetuin Type I, salt essentially freefrom fetal calf serum N-acetylneuraminic acid

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A 9791 Asialoglycophorin Lyophilized powder, Predominantly Asialoglycophorin Afrom human blood Type MN

G 7900 Monoclonal Anti-Glycophorin A Produced in mouse, Ascites fluid, Clone E4, Liquid(a) antibody

G 7650 Monoclonal Anti-Glycophorin A,B Produced in mouse, Clone E3, Ascites fluid, Liquid(a,d) antibody

G 7775 Monoclonal Anti-Glycophorin C Produced in mouse, Clone E5 or 2C10, Whole antiserum, Liquid(b) antibody

B 9277 Monoclonal Anti-Band 3 antibody Produced in mouse, Clone BIII-136, Ascites fluid, Liquid

R 4011 Monoclonal Anti-Red Cell Wrb Produced in mouse, Affinity purified, One unit will bind 1.0 mg of d-biotinAntigen antibody binds to Wrb, a composite blood group antigen resulting from the association between

Glycophorin A and Band 3

A 8706 Avidin Affinity purified, One unit will bind 1.0 mg of d-biotinfrom egg white, recombinant

B 8041 Biglycan Essentially salt-free, lyophilized powder, sterile-filteredfrom bovine articular cartilage Interacts with collagen type I, as well as with fibronectin and TGF-b.

C 1063 Chorionic gonadotropin human Lyophilized powder, sterile-filteredvial of 2,500 I.U.

C 5297 Chorionic gonadotropin human Lyophilized powder, Potency: approx. 3,000 I.U. per mg

G 4877 Gonadotropin 1,500-6,000 I.U./mg, PMSGfrom pregnant mare serum

C 0755 Conalbumin chicken egg white Substantially iron-free, Minimum 98% (Ovotransferrin)

D 8428 Decorin Salt-free, lyophilized powder, sterile-filteredfrom bovine articular cartilage Decorin interacts with collagen type I and II, fibronectin, thrombospondin, and TGF-b.

F 2379 Fetuin Lyophilized powder (from sodium acetate buffer)from fetal calf serum free N-acetylneuraminic acid approx. 0.2%

F 3004 Fetuin Lyophilized powderfrom fetal calf serum Further processing of F 2379 by gel filtration.

F 3879 Fibrinogen Type I, Contains approx. 15% sodium citrate and approx. 25% sodium chloride.from human plasma Approx. 60% protein (80-90% of protein clottable)

F 4385 Fibrinogen Contains approx. 20% sodium citrate and approx. 30% sodium chloride.from murine plasma Approx. 50% protein (over 80% of protein clottable)

F 8630 Fibrinogen Type I-S powder, powder containing approx 75% protein, approx. 10% sodium citrate from bovine plasma and approx. 15% sodium chloride

F 4883 Fibrinogen Essentially plasminogen-free powder containing approx. 50% protein, approx. 20% sodium from human plasma citrate and approx. 30% sodium chloride

H 7017 Hemocyanin Megathura crenulata Lyophilized powder, contains stabilizing buffer, mol wt 8,000-9,000 kDa(keyhole limpet) vial of 20 mg KLH (in approximately 100 mg total weight)

H 8283 Hemocyanin Megathura crenulata in PBS solution, Chromatographically purified(keyhole limpet) Concentration 3-7 mg/ml protein (A280)

K 3009 Keratan sulfate proteoglycan sterile-filteredfrom bovine cornea

L 0520 Lactoferrin approx. 90% (SDS-PAGE) Lyophilized powderfrom human milk Chromatographically purified. Contains sodium chloride

L 2020 Laminin 1 mg/ml in Tris buffered NaCl cell culture, testedfrom Engelbreth-Holm-Swarm sterile-filteredmurine sarcoma

M 1778 Mucin Type III partially purified powder, Bound sialic acids, approx. 1%from porcine stomach

M 3895 Mucin Type I-S Lyophilized powder, Neuraminidase substratefrom bovine submaxillary glands Bound sialic acids approx. 12%

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P 6782 Peroxidase horseradish Essentially salt-free, Lyophilized powder 250-330 units/mg solid (using pyrogallol) Type VI-A

P 8375 Peroxidase horseradish Type VI Essentially salt-free, lyophilized powder 250-330 units/mg solid

P 5661 Plasminogen e-Aminocaproic acid free, Lyophilized powder containing NaCl, EDTA, lysine, and Tris buffer, from human plasma plasmin

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Chemical Deglycosylation Strategies

Analysis of the glycan structure of glycoproteins normally requires enzymatic or chemicalmethods of deglycosylation.

Removal of carbohydrates from glycoproteins is useful for a number of reasons: To simplify analysis of the peptide portion of the glycoprotein To simplify the analysis of the glycan component To remove heterogeneity in glycoproteins for X-ray crystallographic analysis To remove carbohydrate epitopes from antigens To enhance or reduce blood clearance rates of glycoprotein therapeutics To investigate the role of carbohydrates in enzyme activity and solubility To investigate ligand binding For quality control of glycoprotein pharmaceuticals

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R

Hydrazinolysis

Re-N-acetylation

Regeneration ofreducingterminus

OO

O

OC

CC

HO

NH

H3C

CH2OHHN CH2

CHNH

Peptide

O

Peptide

RO

O

OC

HO

NH

H3C

CH2OHHN

NH2

RO

O

OC

HO

NH

H3C

CH2OHHN

NH CH3

O

CR

OO

OC

HO

NH

H3C

CH2OHOH

HydrazinolysisHydrazine hydrolysis has been found to be effective inthe complete release of unreduced O- and N-linkedoligosaccharides. Selective and sequential release ofoligosaccharides can be accomplished by initial mildhydrazinolysis of the O-linked oligosaccharides at 60 Cfollowed by N-linked oligosaccharides at 95 C. Hydrazinehydrolysis leaves the glycan intact but results in destruc-tion of the protein component.

Glycan release is accomplished by the addition of freshanhydrous hydrazine to a salt-free, freshly lyophilizedglycoprotein sample. The volume of hydrazine shouldproduce a protein concentration of 5 to 25 mg/ml. Thereaction mixture should be capped immediately. For therelease of both N- and O-linked glycans incubation shouldbe for 4 hours at 95 C. For the selective release of O-linked glycans, incubation at 60 C for 5 hours is suitable.

Excess unreacted hydrazine can be removed under highvacuum at a temperature not exceeding 25 C. The vacuumpump should be equipped with an activated charcoal/alumina trap. Addition of small aliquots of anhydroustoluene may be required to bring the sample to com-plete dryness.

The dried sample should then be re-N-acetylated on iceby the addition of ice-cold saturated sodium bicarbonatesolution, followed immediately by the addition of aceticanhydride. The sample is mixed gently and incubated atroom temperature for 10 minutes. A second equal aliquotof acetic anhydride is added and incubated for an additional20 minutes. A 5-fold molar excess of acetic anhydrideover the amine content of the protein should be used.The volume of sodium bicarbonate added should yield a 0.5 M acetic anhydride final concentration in the reac-tion mixture.

A small amount of the released glycan pool may exist asthe acetohydrazide derivative. These derivatives may beconverted to the unreduced glycans by resuspension ofthe dryed glycan pool in 1 mM Cu(II) acetate in 1 mMacetic acid and incubation at room temperature for one hour.

Dowex 50WX2 can be used to remove the cations andthe sample is washed through with water.

Glycan and protein components can be separated by gelfiltration or by paper chromatography.

Complete details for this procedure are given in Methodsin Enzymology. Care should be taken during all steps ofthis procedure as many of the reagents and reactions areextremely hazardous and highly reactive.

References1. Patel, T., Bruce, J., Merry, A.H., Bigge, J.C., Wormald, M.R., and

Parekh, R.B., Biochemistry, 32, 679-693 (1992).2. Takasaki, S., and Kobata, A., Meth. Enzymol., 50, 50-54 (1978).

Key Products

H 2761 Hydrazine

24,451-1 Toluene, Anhydrous

S 6297 Sodium Bicarbonate

A 6404 Acetic Anhydride

C 5893 Cupric Acetate

21,747-6 Dowex 50WX2-400

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Alkaline b-EliminationRelease of glycans by alkaline b-elimination utilizes ammo-nium hydroxide/carbonate or sodium hydroxide (in con-junction with sodium borohydride).

O-Glycosidic linkages between glycans and the b-hydroxylgroups of serine or threonine are readily hydrolyzed bydilute alkaline solutions (0.05 to 0.1 M sodium hydroxideor potassium hydroxide) under mild conditions (45 to 60 Cfor 8 to 16 hours) leading to the liberation of O-glycansby the mechanism of b-elimination. To prevent isomeriza-tion or degradation of the carbohydrates by peelingreactions, the hydrolysis is performed in the presence ofa reducing agent (0.8 to 2 M sodium borohydride). Thisresults in the formation of the reduced (alditol) forms ofthe glycans. N-Linked glycans are not cleaved under theseconditions, and neither are O-glycans attached to tyrosine,hydroxyproline, and hydroxylysine. Furthermore, the b-elimination reaction does not take place if the glycan is attached to serine or threonine at the carboxy-terminusof the protein.

For quantitative release of N-linked glycans, harsheralkaline conditions are required (1 M sodium hydroxideat 100 C for 6 to 12 hours). Again, the reaction must beperformed under reducing conditions (1 to 2 M sodiumborohydride) to prevent peeling reactions taking placeon the released N-glycans. N-Acetylglucosamine (GlcNAc)residues are de-acetylated during this reaction and mustbe re-N-acetylated (acetic anhydride in methanol) duringthe recovery of the glycans.

References1. Carlson, D.M., and Blackwell, C., J. Biol. Chem., 243, 616-626 (1968).2. Lloyd, K.O., Burchell, J., Kudryashov, V., Yin, B.W.T., and Taylor-

Papadimitriou, J., J. Biol. Chem., 271, 33325-33334 (1996).3. Morelle, W., Guyetant, R., and Strecker, G., Carbohydr. Res., 306,

435-443 (1998).

Trifluoromethanesulfonic AcidTrifluoromethanesulfonic acid (TFMS) hydrolysis leaves anintact protein component, but results in destruction ofthe glycan.

Glycoproteins from animals, plants, fungi, and bacteriahave been deglycosylated by this procedure. It has beenreported that the biological, immunological, and receptorbinding properties of some glycoproteins are retainedafter deglycosylation by this procedure, although thismay not be true for all glycoproteins. The reaction isnon-specific, removing all types of glycans, regardless ofstructure, although prolonged incubation is required forcomplete removal of O-linked glycans. Also, the inner-most Asn-linked GlcNAc residue of N-linked glycansremains attached to the protein. The method removesthe N-glycans of plant glycoproteins that are usuallyresistant to enzymatic hydrolysis.

References1. Patel, T.P., and Rarekh, R.B., Meth. Enzymol., 230, 57-66 (1994).2. Bendiak, B. and Cumming, D.A., Carbohydr. Res., 151, 89 (1986).3. Makino, Y., et al., J. Biochem., 128, 175-180 (2000).4. Piller, F., and Piller, V., in Glycobiology: A Practical Approach,

Fukuda, M. and Kobata, A. (Eds), pp. 291-328 (IRL/Oxford Univ.Press, (Oxford UK 1993)).

5. Burgess, A.J., and Norman, R.I., Eur. J. Biochem. 178, 527-533(1988).

6. Edge, A.S.B., J. Biochem., 376, 339-350 (2003).7. Sojar, H.T., and Bahl, O.P., Meth. Enzymol, 138, 41-350 (1987).8. Edge, A.S.B., et al., Anal. Biochem., 118, 131-137 (1981).


GlycoProfile IV ChemicalDeglycosylation KitProduct Code PP0510

The GlycoProfile ChemicalDeglycosylation Kit has been opti-

mized to provide a rapid, conven-ient, and reproducible method to

remove glycans from glycoproteins byreaction with trifluoromethanesulfonic acid

(TFMS). The deglycosylated protein can then be recoveredusing a suitable downstream processing method. The kitcontains sufficient reagents and a glycoprotein standard,for a minimum of 10 reactions when the sample size isbetween 1 and 2 mg of a typical glycoprotein. Unlike otherchemical deglycosylation methods, hydrolysis with anhy-drous TFMS is very effective at removing O- and N-linkedglycans (except the innermost Asn-linked GlcNAc orGalNAc) and results in minimal protein degradation.

Visit

sigma-aldrich.com/glycogo

to find out more about the latest

glycobiology products

from Sigma-Aldrich

COMINGSOON!

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Enzymatic Deglycosylation Strategies

Sequential hydrolysis of individual monosaccharides from glycans can be useful for theelucidation of the structure and function of the glycan component. Due to the restraintsof the specificity of glycolytic enzymes currently available, sequential hydrolysis of individualmonosaccharides is also required in many instances in order to completely remove a glycancomponent enzymatically. This is particularly true in the enzymatic deglycosylation ofmany O-linked glycans.

N-Linked Glycan StrategiesUse of the enzyme PNGase F is the most effective methodof removing virtually all N-linked oligosaccharides fromglycoproteins. The oligosaccharide is left intact and, there-fore, suitable for further analysis (the asparagine residuefrom which the sugar was removed is deaminated toaspartic acid, the only modification to the protein). Atripeptide with the oligosaccharide-linked asparagine asthe central residue is the minimal substrate for PNGase F.

However, oligosaccharides containing a fucose a(1-3)-linkedto the asparagine-linked N-acetylglucosamine, commonlyfound in glycoproteins from plants or parasitic worms,are resistant to PNGase F. N-Glycosidase A (PNGase A),isolated from almond meal, must be used in this situation.This enzyme, however, is ineffective when sialic acid ispresent on the N-linked oligosaccharide. Other commonly

used endoglycosidases such as Endoglycosidase H and the Endoglycosidase F series are not suitable for generaldeglycosylation of N-linked sugars because of their limitedspecificities and because they leave one N-acetylglucosamineresidue attached to the asparagine.

Steric hindrance slows or inhibits the action of PNGase Fon certain residues of glycoproteins. Denaturation of theglycoprotein by heating with SDS and 2-mercaptoethanolgreatly increases the rate of deglycosylation.

Through sequential deglycosylation of monosaccharides,all complex oligosaccharides can be reduced to the triman-nosyldiacetylchitobiose core by selective hydrolysis with a neuraminidase, b-galactosidase, and N-acetylglucosa-minidase, available as part of the Enzymatic DeglycosylationKit (Product Code E-DEGLY). Fucosidases may be requiredin some situations.

PNGase F

R = N- and C-substitution by groups other than HR = H or the rest of an oligosaccharide R3 = H or a(1-6)fucose

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Native and Sequential N-LinkedGlycan StrategiesFor some glycoproteins, no cleavage by PNGase F occursunless the protein is denatured. For others, some or allof the oligosaccharides can be removed from the nativeprotein after extensive incubation of three days or longer.PNGase F will remain active under reaction conditions for at least three days allowing extended incubations ofnative glycoproteins. In general, it appears that particularresidues, due to their location in the native protein struc-ture, are resistant to PNGase F and can not be removedunless the protein is denatured.

Endoglycosidases F1, F2, and F3 are less sensitive to proteinconformation than PNGase F and are more suitable fordeglycosylation of native proteins. Sigmas Native ProteinDeglycosylation Kit (Product Code N-DEGLY) supplies all

three of these enzymes with reaction buffers and detailedinteractions. The linkage specificities of EndoglycosidasesF1, F2, and F3 suggest a general strategy for deglycosyla-tion of proteins that may remove all classes of N-linkedoligosaccharides without denaturing the protein. Initiallycomplex oligosaccharides can be reduced to the trimanno-syldiacetylchitobiose using neuraminidase, b-galactosidase,and N-acetylglucosaminidase. Fucosidases may berequired in some situations. The remaining trimannosyl-diacetylchitobiose core structures can be removed withEndoglycosidase F3. Bi- and triantennary structures canbe immediately removed by Endoglycosidases F2 and F3, respectively.

High-mannose (oligomannose) and hybrid structures canbe removed by Endoglycosidase F1, but not complexoligosaccharides.

High-Mannose Glycans

Hybrid Glycans

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Biantennary Glycans

Endo F2 and Endo F3 have the ability to cleave complexstructures. Endo F2 cleaves biantennary complex and tolesser extent high mannose oligosaccharides. Fucosylationhas little effect on Endo F2 cleavage of biantennarystructures. Endo F2 will not cleave hybrid structures.Endo F3 cleaves biantennary and triantennary complexoligosaccharides. However, non-fucosylated biantennaryand triantennary structures are hydrolyzed at a slow rateby Endo F3. Core fucosylated biantennary structures areefficient substrates for Endo F3 oligosaccharides. Core

fucosylation of biantennary structures increases activityup to 400-fold. Endo F3 has no activity on oligomannoseand hybrid molecules.

Endo F3 will cleave fucosylated and non-fucosylated triman-nosyl core structures on free and protein-linked glycans.Native deglycosylation of complex tetraantennary glycansrequires sequential hydrolysis down to the trimannosyl-diacetylchitobiose core.

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Triantennary Glycans


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Complex Tetraantennary Glycans


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Di- and Trisialated O-Linked Glycans

O-Linked Core 2 Hexasaccharide


O-Linked Glycan StrategiesThere is no enzyme comparable to PNGase F for removingintact O-linked sugars. Monosaccharides must be sequen-tially hydrolyzed by a series of exoglycosidases until onlythe Gal-b(1-3)-GalNAc core remains. O-Glycosidase canthen remove the core structure intact with no modifica-tion of the serine or threonine residue. Denaturation ofthe glycoprotein does not appear to significantly enhanceO-deglycosylation. Any modification of the core structurecan block the action of O-Glycosidase. The most commonmodification of the core Gal-b(1-3)-GalNAc is a mono-, di-,or tri-sialylation. These residues are easily removed by a(2-3,6,8,9)-Neuraminidase since only this enzyme is capa-ble of efficient cleavage of the NeuNAc-a(2-8)-NeuNAcbond. Another commonly occurring O-linked hexasaccharidestructure contains b(1-4)-linked galactose and b(1-6)-linkedN-acetylglucosamine as well as sialic acid. Hydrolysis of

this glycan will require, in addition to neuraminidase, ab(1-4)-specific galactosidase and an N-acetylglucosaminidase.A non-specific galactosidase will hydrolyze b(1-3)-galactosefrom the core glycan and leave O-linked GalNAc that cannotbe removed by O-Glycosidase. b(1-4)-Galactosidase and b-N-acetylglucosaminidase can be used for the hydrolysisof these and any other O-linked structures containingb(1-4)-linked galactose or b-linked N-acetylglucosaminesuch as polylactosamine. Less common modifications thathave been found on O-linked oligosaccharides include a-linked galactose and a-linked fucose. Directly O-linkedN-acetylglucosamine (found on nuclear proteins) and a-linked N-acetylgalactosamine (found in mucins) havealso been reported. Additional exoglycosidases are neces-sary for complete O-deglycosylation when these residuesare present. Fucose and mannose directly O-linked toproteins cannot presently be removed enzymatically.

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R1 = N- and C-substitution by groups other than HR2 = H or the rest of an oligosaccharide structureR3 = H or a(1-6)fucose

Deglycosylation KitsGlycoProfile IEnzymatic In-Gel N-Deglycosylation KitProduct Code PP0200

Glycosylation often leads to problems in subsequent pro-tein analysis procedures. Glycopeptides generally do notreadily ionize during MS analysis leading to insufficientspectral data. Furthermore, proteolytic digestion of thenative glycoprotein is often incomplete due to sterichindrance by the oligosaccharides. Removal of thecarbohydrate groups from a glycoprotein prior toprotein identification is preferred.

Sigmas GlycoProfile I Enzymatic In-gel N-DeglycosylationKit is optimized to provide a convenient and reproduciblemethod to N-deglycosylate and tryptically digest proteinsamples in 1D or 2D polyacrylamide gel slices for subse-quent MS or HPLC analysis. The procedure is suitable forCoomassie Blue and Colloidal Coomassie stained gels andmay be used with gels silver stained and destained usingSigmas Proteo Silver Plus Kit (Product Code PROT-SIL2).GlycoProfile Enzymatic In-gel N-Deglycosylation kit includesthe enzymes and reagents necessary for N-linked degly-cosylation and tryptic digestion. The samples can then bedesalted and concentrated for analysis by MALDI-TOF orelectrospray MS.

Features & Benefits Provides all components for in-gel deglycosylation and

trypsinization of protein samples Conveniently pre-pares deglycosylated protein samples for analysis by MS or HPLC

Utilizes PNGase F for the enzymatic removal of N-linkedglycans Proteins remain intact, unlike the use of chem-ical deglycosylation which can degrade the protein

Includes Proteomics Grade PNGase F and Trypsin Highly purified enzymes possess no unwanted activitiesor additives to complicate analysis

PNGase F is supplied lyophilized from a low salt buffer Allows reconstitution of the enzyme to any concentra-tion needed

Works in solution or with gel slices Allows choice of methods

Components

Destaining Solution

Proteomics Grade PNGase F

Proteomics Grade Trypsin

Trypsin Solubilization Reagent

Trypsin Reaction Buffer

Invertase (positive control)

Peptide Extraction Solution

Biotech Grade Acetonitrile

In-gel Deglycosylation

Excise protein of interest

Add water

Destain gel slice

Place gel slice in siliconized tube

Remove destaining solution and dry gel in Speed Vac

Rehydrate with PNGase F solution

Remove glycan- containing supernatant.Analyze if desired.

Remove and discard liquid

Wash gel slice with water

Dry gel slice in Speed Vac

Incubate for 30 min. at 37 C

Incubate 2 hours toovernight at 37 C

Sample is now ready to be rehydrated in Trypsin solution (see Trypsin In-Gel Digest kit).


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GlycoProfile IIEnzymatic In-Solution N-Deglycosylation KitProduct Code PP0201

The GlycoProfile Enzymatic In-Solution N-DeglycosylationKit has been optimized to provide a convenient and repro-ducible method to remove N-linked glycans from glyco-proteins and is compatible with subsequent MALDI-TOFmass spectrometric analysis without interference fromany of the reaction components. The kit contains suffi-cient enzyme, glycoprotein standard and reagents, for a minimum of 20 reactions when the sample size isbetween one to two mg of a typical glycoprotein.

Features & Benefits Provides all components for in-solution N-linked degly-

cosylation of protein samples Conveniently preparesdeglycosylated protein samples for analysis by MS,HPLC and PAGE

Reagents are optimized for direct MS analysis No needfor post-reaction sample clean up

Utilizes PNGase F for the enzymatic removal of N-linkedglycans Proteins remain intact, unlike the use of chem-ical deglycosylation which can degrade the protein

Includes Proteomics Grade PNGase F and Trypsin Highly purified enzymes possess no unwanted activitiesor additives to complicate analysis

PNGase F is supplied lyophilized from a low salt buffer Allows reconstitution of the enzyme to any concentra-tion needed

Components

Proteomics Grade PNGase F

Ribonuclease B

10x Reaction Buffer

Octyl b-D-Glucopyranoside

2-Mercaptoethanol


In-solution Deglycosylation

Add denaturant

Aliquot glycoprotein sample

Incubate at 100 C for 10 min

Incubate at 37 C for 1 hour

Analyze by MS, PAGE or HPLC

Cool to room temperature

Add reaction buffer and PNGase F


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Key EndoglycosidasesPNGase F Synonyms: Glycopeptidase F; N-Glycosidase F; Peptide N-Glycosidase F; Peptide-N4-(acetyl-b-glucosaminyl)-asparagine amidase

EC 3.5.1.52 Molecular Weight: 36 kDa

PNGase F cleaves all asparagine-linked complex, hybrid,or high mannose oligosaccharides unless b(1-3) corefucosylated. The asparagine must be peptide bonded at both termini. The asparagine residue from which theglycan is removed is deaminated to aspartic acid.

Detergent and heat denaturation increases the rate ofcleavage up to 100 times. Most native proteins can stillbe completely N-deglycosylated, but incubation time mustbe increased. The optimal pH is 8.6 and the enzyme isactive in the pH range of 6 to 10.


R PNGase F

Aspartic Acid Residue

OO

O

OC

CC

HO

NH

H3C

CH2OHHN CH2

CHNH

Peptide

O

Peptide

RO

O

OC

HO

NH

H3C

CH2OH

OH

Asparagine ResidueCore GlcNAcOC

CHO

CH2CH

NH

Peptide

O

Peptide

+

SDS-PAGE analysis of RNase B treated with varying amounts of PNGase F Enzyme

PNGase F Products

P 7367 PNGase F Synonyms: N-Glycanase, N-Glycosidase F, Proteomics Grade8

Features and Benefits Excellent for applications requiring N-linked deglycosylation. Superior performance in on-blot, in-gel, and in-solution digestion methods. High activity, minimum 25,000 units per milligram. Compatible for use in MALDI-TOF Mass Spectroscopy. Each lot tested for suitability.

Proteomics Grade PNGase F from Sigma-Aldrich is extensively purified and contains minimal residual buffer salts and no glycerol or other stabilizers or preservatives that may interfere in sensitive glycoprotein analysis methods.

Lane 1: Control (no PNGase F)Lane 2-10: Test (+ 0.01 to 0.09 U PNGase F)

G 5166 PNGase F Synonyms: N-Glycanase, N-Glycosidase F8 from Chryseobacterium (Flavobacterium), Buffered Aqueous Solution

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PNGase APNGase A (Glycopeptidase A, Peptide N-Glycosidase A),hydrolyzes oligosaccharides containing a fucose a(1-3)-linked to the asparagine-linked N-acetylglucosamine,commonly found in glycoproteins from plants or parasiticworms. These types of glycans are resistant to PNGase F.Like PNGase F the asparagine asparagine residue fromwhich the glycan is removed is deaminated to asparticacid. However, it is ineffective when sialic acid is presenton the N-linked oligosaccharide.


Deglycosylation Products

P 9120 PNGase F Synonyms: N-Glycanase, N-Glycosidase F8 Recombinant, Solution

N 3786 a(23,6,8,9) Neuraminidase Synonyms: Sialidase, Acyl-neuraminyl Hydrolase, Proteomics Gradefrom Arthrobacter ureafaciens

Features and Benefits Compatible for use in MALDI-TOP MS applications. Cleaves all non-reducing, unbranched NANA/NeuNAc and NeuGc residues.

Ideal for complete, non-specific removal of sialic acid groups.

PP0200* Glycoprofile I Excellent performance when used for in-gel N-linked deglycosylation of glycoproteins 8 In-Gel N-Deglycosylation Kit and glycopeptides. Utilizes extensively purified Proteomics Grade PNGase F and Trypsin.

Compatible with MS (TOF or Electrospray) analysis following desalting. Sufficient for a minimum of 10 samples.

PP0201* Glycoprofile II Superior, reproducible method to remove N-linked glycans from glycoproteins. Utilizes 8 In-Solution N-Deglycosylation Kit extensively purified Proteomics Grade PNGase F. Compatible with subsequent MALDI-TOF

mass spectrometric analysis, no need for desalting. Sufficient for a minimum of 20 reactions.

N-DEGLY Native Deglycosylation Kit The N-DEGLY kit includes Endoglycosidase F1, F2, and F3 as well as an optimized reactionbuffer for each. The kit is intended for use in the deglycosylation of N-linked oligosaccharidesfrom glycoproteins under native conditions. Particular residues, due to their location in the native protein structure, are often resistant to the traditional deglycosylation methodsusing PNGase F and cannot be removed unless the protein is denatured. EndoglycosidasesF1, F2, and F3 are less sensitive to protein conformation than PNGase F and are more suit-able for deglycosylation of native proteins.

*For additional product information, refer to pages 30 and 31.

Deglycosylation Products

G 0535 PNGase A Buffered solution, min. 0.5 units/ml

G 1163 O-Glycosidase O-Glycosidase hydrolyzes the serine- or threonine-linked unsubstituted O-Glycan core [Gal-b(1-3)-GalNAc]. Any modification of the core structure can block the action of O-Glycosidase.

E-DEGLY Enzymatic Deglycosylation Kit Completely removes all N- and simple O-linked carbohydrates from glycoproteins.Efficiently digest polysialylated carbohydrates. Simplifies amino acid sequencing applications.Removes carbohydrate epitopes from antigens. Native & denaturing procedures included in bulletin.

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Endoglycosidase F1, F2 & F3Endoglycosidase F1 cleaves between the two N-acetyl-glucosamine residues in the N-linked diacetylchitobioseglycan core of the oligosaccharide, generating a trun-cated sugar molecule with one N-acetylglucosamineresidue remaining on the asparagine. High mannose(oligomannose) and hybrid structures can be removed by Endoglycosidase F1, but not complex, oligosaccharides.Useful under native or non-denaturing deglycosylationconditions.

R1 = Asn or HR2 = Oligomannose or hybrid configurationsR3 = H or a(1-6)fucose

Endoglycosidase F2 cleaves between the two N-acetyl-glucosamine residues in the N-linked diacetylchitobioseglycan core of the oligosaccharide, generating a trun-cated sugar molecule with one N-acetylglucosamineresidue remaining on the asparagine. Endo F2 cleavesbiantennary complex and to a lesser extent high man-nose oligosaccharides. Fucosylation has little effect onEndo F2 cleavage of biantennary structures. Endo F2 willnot cleave hybrid structures. Useful under native or non-denaturing deglycosylation conditions.

R1 = H or AsnR2 = Biantennary and oligomannose configurationsR3 = H or a(1-6)fucose

Endoglycosidase F3 cleaves between the two N-acetyl-glucosamine residues in the N-linked diacetylchitobioseglycan core of the oligosaccharide, generating a trun-cated sugar molecule with one N-acetylglucosamineresidue remaining on the asparagine. Endo F3 cleavesnon-fucosylated biantennary and triantennary structuresat a slow rate, but only if peptide-linked. Core fucosyla-tion of biantennary structures increases activity up to400-fold. Endo F3 has no activity on oligomannose andhybrid molecules. Endo F3 will also cleave fucosylatedtrimannosyl core structures on free and protein-linkedoligosaccharides. Native deglycosylation of complextetrantennary glycans requires sequential hydrolysisdown to the trimannosyl-diacetylchitobiose core.

R1 = N- and C-substitution by groups other than HR2 = Biantennary and triantennary complex oligosaccharides or

trimannosylchitobiose coreR3 = H or a(1-6)fucoseR4 = Asn (H or Asn if core fucosylated)


Endoglycosidase F Products

E 9762 Endoglycosidase F1 Solution, from Chryseobacterium (Flavobacterium) meningosepticum



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Endoglycosidase HEndoglycosidase H cleaves between the N-acetylglucosa-mine residues of the chitobiose core of N-linked glycans,leaving one N-acetylglucosamine residue attached to theasparagine. The specificity of this enzyme is such thatoligomannose and most hybrid types of glycans, including

those that have a fucose residue attached to the corestructure, are cleaved whereas complex type glycans arenot released. Thus this enzyme is extremely useful forselective release of oligomannose or hybrid type glycansfrom glycoproteins. The enzyme is also active againstdolichol-linked glycans containing these structures.

R1 = Oligomannose (2-150)R2 = H or mono- or oligosaccharide at the C2 or C4 positionR3 = H or a(1-6)fucoseR4 = Asn or Dolichol pyrophosphate

The enzyme has a molecular weight of approximately 27 kDa. The workable pH range is between 5.0 and 6.0,with the optimal pH at 5.5. No loss of activity is observedduring incubation at 37 C for 48 hours over the pHrange 4.5 to 8.5. However, below pH 4.5, activity israpidly lost.


Endoglycosidase H Products

A 0810 Endoglycosidase H From recombinant with reaction buffer

E 6878 Endoglycosidase H From Streptomyces griseus

E 7642 Endoglycosidase H Recombinant, from Streptomyces plicatus, expressed in E. coli

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Glycan Analysis and Labeling

In contrast to proteins and peptides, glycansdo not absorb ultraviolet (UV) light strongly,thereby giving a weak detector signal, evenat 214 nm. Furthermore, as glycans withvarious different structures may be presentin minute amounts in glycoprotein hydro-lysates, their detection by UV absorbancemay not be practical. As a result a widerange of alternative techniques have beendeveloped for the detection of glycans.

Instead of UV detectors for on-line HPLCsystem, refractive index and pulsed amper-ometric detectors, with sensitivity in therange of 10 to 100 pmol, have enabledimproved detection of native mono- andoligosaccharides. Other approaches toimprove detection include labeling glycanswith radioactive isotopes, but this tech-nique is not widely used due to the safetyrequirements for the handling of radio-labeled substances.

Most glycoproteins exist as a heterogeneouspopulation of glycoforms or glycosylatedvariants with a single protein backbone anda heterogeneous population of glycans at

each glycosylation site. It has been reportedin the literature that for some glycoproteins,100 or more glycoforms exist at each glyco-sylation site. In view of this heterogeneityand the presence of branched structures,the analysis of glycans is much more com-plicated than protein chemistry. It requiresseveral different strategies to separate andstudy the structure of each individual glycan.

Once the glycans have been released fromthe glycoprotein, the glycan pool can beanalyzed by MALDI-TOF mass spectrometryor, after fluorescent labeling, by either HPLCor MS, or both. This strategy can provide a glycan profile or a glycosylation pat-tern that is highly characteristic of theglycoprotein. The technology can be appliedto compare glycan profiles of glycoproteinsfound in normal and diseased states, or tocompare different batches of recombinantprotein products. Both these techniquesprovide valuable information in terms ofcomposition, linkage and arm specificity(using various exoglycosidases) from whichstructural information on individual glycanscan be elucidated.

Chemical derivatization is now the most common methodused for labeling glycans at their reducing ends by reduc-tive amination. A single molecule of fluorescent label canbe incorporated to each mono- or oligosaccharide, thusallowing molar quantities to be determined. The sensitivity

of detection by this technique is in the low femtomolerange and depending upon the analytical technique, anappropriate fluorescent label can be used. The table belowlists examples of some of the fluorophores widely used in glycan analysis.

Technique Fluorescent Label Abbreviation Product Code

HPLC 2-Aminobenzoic acid 2-AA 10678

2-Aminobenzamide 2-AB 10710

2-Aminopyridine 2-AP 09340

Gel Electrophoresis 8-Aminonaphthalene-1,3,6-trisulfonic acid ANTS 08658

2-Aminoacridone AMAC 06627

Capillary Electrophoresis 9-Aminopyrene-1,3,6-trisulfonic acid APTS 09341

Mass spectrometry 2-Aminobenzamide 2-AB 10710

Glycan Labeling

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HPLC Analysis of GlycansThe released glycan pool from glycoprotein hydrolysiscan be separated into components depending upon thecharacteristics of the glycans and the HPLC matrices used.Three types of chromatography, normal phase, weak anionexchange, and reversed phase HPLC, have been generallyused with fluorescent-labeled oligosaccharides for theseparation of sialylated and neutral N- and O-linkedglycans. High pH anion exchange chromatography withpulsed amperometric detector can be used with nativeglycans, but has a major drawback in that the separationis achieved in high molarity sodium hydroxide that has to be removed to recover glycans for further structuralanalysis.

Normal phase chromatographyThe column used in this mode of chromatography consistsof a matrix with imide or amide functional groups. Samplesare applied in an organic solvent solution to the columnare then eluted with increasing concentration of aqueousbuffer. This exploits the subtle differences in hydrophilicitybetween individual glycans and their affinity for thecolumn matrix, thereby achieving high resolution andreproducibility. The ability to analyze sialylated and neutralsugars in one chromatographic run makes it a useful tech-nique for profiling both N- and O-glycan pools, and formaking comparisons between different glycan samples.Figures 1 and 2 show the elution profiles of N-linkedglycans released from two model glycoproteins, RNase Band Fetuin, respectively.

Figure 1. HPLC Profile of the 2-AB labeled N-linked glycan libraryobtained from RNase B.

Figure 2. HPLC Profile of the 2-AB labeled N-linked glycan libraryobtained from Fetuin.

Partially hydrolyzed dextran, as a source of glucose oligo-mers, is used as an external standard and the elutionposition of each peak is expressed in glucose units (gu)(see Fig. 3). The elution positions of peaks in an unknownglycan pool are assigned an overall gu value by compari-son with the standard dextran ladder. These values maythen be used to predict possible structures for unknownglycans. For example, it has been reported that the glycansreleased from RNase B consist solely of N-linked oligo-mannose structures. The five peaks obtained upon profilingof the glycans from RNase B can therefore be assignedstructures ranging from Man-5 to Man-9, as indicated on Figure 1.

Figure 3. Separation of partially hydrolyzed 2-AB labeled dextran onnormal phase HPLC. The numbers indicate glucose units (gu).

3,300

4,400

2,200

1,100

0

0 20

1 2 3 45

6

7

8

910

11

40 60 80 100 120 140 160

Retention time (minutes)

Flu

ore

scen

ce

20,000

12,000

16,000

8,000

4,000

0

70 80 90 100 110 120 130

Fetuin


Flu

ore

scen

ce

800

600

400

200

0

70 80 90 100 110 120

Man-6

Man-7

Man-8

Man-9

Man-5

RNase B


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Weak anion exchange chromatographyWeak anion-exchange chromatography relies upon therelative binding affinities of the glycans to the weak anionexchange matrix in the column. This type of chroma-tography separates N- and O-linked glycans on the basis ofthe number of charge groups they contain. The elutionposition is therefore determined by the number of sialicacid, sulfate, phosphate, or uronic acid moieties presenton the glycan, but also to some extent by the size of theglycan. Thus, within one charge band, larger structureselute before smaller ones. For example, Figure 4(A) showsthe separation of the N-linked glycan library releasedfrom fetuin into five major peaks. The flow-through peakcontains neutral glycans, while peaks eluting with increas-ing time represent the mono-, di-, tri- and tetrasialylatedglycans of fetuin.

In some of these charge classes, for example in the disialylated glycan peak area shown in Figure 4(A), thepartially-resolved peak contains bi- and triantennarystructures with the triantennary glycans eluting first. The peaks corresponding to each charge band can becollected, concentrated, further separated, and analyzedby normal phase chromatography. Two examples areshown in Figures 4(B) and 4(C).

A)

B)

C)

Figure 4. Separation of neutral and acidic glycans of fetuin by weakanion-exchange chromatography. (A) The separation of the glycanpool into neutral, mono-, di-, tri- and tetrasialylated glycans. (B) Further separation of the neutral glycan fraction from (A) bynormal phase chromatography. (C) Further separation of the mono-sialylated glycan fraction from (A) by normal phase chromatography.

Reversed phase chromatographyReversed phase chromatography separates sugars on thebasis of hydrophobicity and can be carried out on a C18column. This method is complementary to the normal phasechromatography, allowing some glycans that co-elute on one system to be resolved by the other system. Thisapproach is important during structure determination ofthe glycan to assess the shift in the retention time upontreatment with a particular exoglycosidase. The sample isapplied in aqueous buffer to the column and eluted withincreasing concentration of organic solvent. Unlike thenormal phase chromatography, the elution positions ofglycans are measured by comparison with an arabinoseladder and assigned as arabinose units (au).

300

400

200

100

0

60 70 80 90 100 110 120 130 140


Flu

ore

scen

ce

Monosialylated glycans

300

400

200

100

0

60 70 80 90 100 110 120 130 140


Flu

ore

scen

ce

Neutral glycans

6,000

8,000

4,000

2,000

00 2

TetraTriDi

4 6 8 10 12 14 16 18 20 22


Flu

ore

scen

ce

Mono

Neutral

Fetuin glycanlibrary

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Mass Spectrometry of GlycansThe development of modern mass spectrometry (MS)equipment with high resolution and mass accuracy hasled to its use in analyzing glycans for both profiling andstructural studies. Unlike HPLC methods, a substantiallylarger amount, about 10-20 times, of glycan is normallyrequired for a single MS spectrum. Matrix-assisted laserdesorption/ionization time-of-flight (MALDI-TOF) massspectrometry is the most widely used MS technique. Theinformation gained on using MALDI MS is mass weightand this can be used to assign putative monosaccharidestructures present in a pure oligosaccharide since the massof a monosaccharide is measured with a high degree ofaccuracy. Like HPLC, MALDI MS can also be used in con-junction with exoglycosidase enzymes for structural andlinkage analysis of glycans.

Different approaches and matrices are used for the analy-sis of neutral and acidic glycans (those that contain sialicacid) by MALDI MS. Neutral glycans ionize with reasonableefficiency using 2,5-dihydroxybenzoic acid (2,5-DHB)(Product Code D 1942) as the matrix in positive ion mode.Acidic glycans generally provide poor MALDI spectra withDHB matrices due to variable losses of sialic acids or car-boxyl groups leading to multiple peaks. These glycansare therefore analyzed in negative ion mode using analternative matrix.

MS of neutral glycansUnlike peptides, neutral glycans exhibit low ionizationefficiency and the [M+H]+ ion is therefore not sufficientlyabundant. However, these glycans can be detected asalkali metal adducts which ionize efficiently. Normally,[M+Na]+ is the major ion, accompanied by a weaker [M+K]+

ion. Other adducts, such as [M+Li]+ can be generated bythe addition of the appropriate inorganic salt to the matrix.The inclusion of NaCl in the matrix solvent allows theglycans to ionize predominantly in the [M+Na]+ formwith little or no [M+K]+ ion formation.

Normally, use of the 2,5-DHB matrix is sufficient for mostapplications. However, it has been reported that, in someinstances, a 2-3 fold increase in sensitivity can be obtainedwith the use of Super 2,5-DHB matrix as it allows forsofter desorption with a reduction in metastable ionformation. Super 2,5-DHB consists of a mixture (90:10ratio, wt %) of 2,5-DHB and 2-hydroxy-5-methoxybenzoicacid, respectively.

Ovalbumin (0.5 mg) was incubated with 7 units of PNGase Fenzyme (Product Code P 7367) at 37 C for 3 hours andthe glycans recovered by ultrafiltration. A third of thismaterial was dried under vacuum centrifugation andthen analyzed by MALDI-TOF MS. Figure 5 shows the N-linked glycan profile of ovalbumin.

100

%

0800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 2200 2300

975.16

990.12

991.141035.10

B1095.08

974.16

A933.20

C1136.22

1152.26

D1257.29

1298.30

E1339.30

1419.33

1501.41

F1542.41

1558.40

G1663.48

1704.52

H1745.52

1762.53

I1866.59

1907.65

J1948.57

1964.66K

2110.68 L2151.74

m/z

Figure 5. Positive ion MALDI MS of the N-linked glycans from oval-bumin released by treatment with PNGase F. Recorded from 2,5-DHB

as the matrix. The letters indicate identified peaks, given on thenext pages.

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A

B

C

Refer to Structure Key on pages 2-4

D

E

F


Corresponding Glycan Structures for MS Profile

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Refer to Structure Key on pages 2-4

J

K

L


Corresponding Glycan Structures for MS Profile

A)

B)

1715 1720 1725 1730 1735 1740 1745 1750 1755 1760 1765 1770 1775m/z0

100

%

1743.58

1744.59

1745.59

1746.59

1747.591760.56

1748.59

915 920 925 930 935 940 945 950 955m/z0

100

%

933.32

931.11915.16

934.32

935.32

936.33

949.29937.33 950.29

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ProcedureThe following method, commonly referred to as thedried-droplet method, is based on the original MALDIexperiments and remains the most commonly usedmethod in the mass spectrometry community.

1. Transfer 3 ml of the appropriate matrix solution (either DHB or Super DHB at 10 mg/ml) into a 0.5 mlEppendorf tube.

2. Add 1 ml of 10 pmol/ml solution of a standard glycan oran unknown sample solution to the tube containingthe matrix. Vortex to mix.

3. Dispense an aliquot (approximately 1.5 ml) of thismixture onto the MALDI target plate.

4. Allow to dry at room temperature. Note: Mass spectra can be acquired at this stage.However the following steps have been reported toimprove signals by forming an even film of crystals,which reduces the need to search for sweet spots on the target.

5. Onto the dried spot, dispense 1 ml of ethanol andallow the matrix plus analyte mixture to recrystallizeon the target plate.

6. Dry the spot. The target is now ready to acquire mass spectra.

7. Figure 6 shows representative MALDI-TOF MS tracesobtained for three glycan standards Man-3 glycan,Man-8 glycan, and NA4 glycan.

Glycan Analysis: General MS Protocol

C)

Figure 6. MALDI-TOF mass spectra of (A) Man-3 glycan, (B) Man-8 glycan, and (C) NA4 glycan obtained in positive ion reflectron mode usingDHB as matrix. Each of these glycans are detected as the so

Documents

Glycosylation Methods and Analysis Sigma