determining the structure of unit 12.6 oligosaccharides n- and o-linked to glycoproteins

8/11/2019 Determining the Structure of UNIT 12.6 Oligosaccharides N- and O-Linked to Glycoproteins

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UNIT 12.6Determining the Structure ofOligosaccharides N- and O-Linked toGlycoproteins

Many proteins involved in biological events are glycosylated. A glycoprotein consists of

a mixture of glycosylation variants of a single polypeptide chain, known as glycoforms. It

has become clear that a detailed understanding of the roles that glycosylation plays in the

biosynthesis, transport, biological function, and degradation of a glycoprotein can only beachieved when the protein and sugar(s) are viewed as an entity. Many glycoproteins can

now be modeled by combining glycan sequencing data and oligosaccharide structural

information with protein structural data. Pivotal to this approach is sensitive, state-

of-the-art oligosaccharide sequencing technology, which can give a rapid insight into

the glycosylation of a glycoprotein without the need for sophisticated equipment and

expertise.

To this end the strategy described at this unit has been developed at the Glycobiology

Institute, Oxford, where it is in daily use. These protocols enable the glycosylation of 5

to 30g of glycoprotein to be analyzed either directly from SDS-PAGE gels (N-linked

sugars) or from 10 g to 2 mg of glycoprotein following hydrazinolysis for N- and/or

O-linked sugars. The strategy is based on high-performance liquid chromatographic(HPLC) technology, which can be used alone or combined with mass spectrometric

techniques (UNIT 12.7). Using HPLC or mass spectrometry (MS), the main characteristics

of the oligosaccharides in a glycan pool can be determined within a few days. The

preparative nature of HPLC allows individual sugars to be isolated for more detailed

analysis if this is required.

These advances open up the possibility of ranking high-throughput oligosaccharide

sequencing alongside that of proteins and DNA. The increasing efficiency of the meth-

ods used to release the glycans and the improved sensitivity of the detection systems

suggest that it will soon be possible to characterize oligosaccharides directly from two-

dimensional gels used in proteomics.

GLYCAN ANALYSIS COMPLEMENTS PROTEIN STRUCTURAL ANALYSISTO GIVE A MORE COMPLETE VIEW OF A GLYCOPROTEIN

Many proteins involved in major biological events such as reproduction, immune defense,

cell-cell interaction the fibrinolytic pathway, and remodeling of the extracellular matrix

are glycosylated, and the importance of the attached sugars for the function of these

molecules is now well established (Varki, 1993; Rudd and Dwek, 1997a; Butler et al.,

2003; Ko et al., 2003; Merry et al., 2003; Peracaula et al., 2003; Scanlan et al., 2003;

Rudd et al., 2004). Whereas the synthesis of the polypeptide chain of a glycoprotein is

under genetic control, the oligosaccharides are attached to the protein and processed by

a series of enzyme reactions without the rigid direction of nucleic acid templates.

N-glycosylation is a cotranslational modification available to, but not necessarily used

by, all proteins that contain the sequon AsnXSer/Thr and occasionally Cys (where X is

any amino acid except Pro) in their primary sequence. As the nascent protein enters the

endoplasmic reticulum (ER) a block of sugars known as the oligosaccharide precursor

(Glc3Man9GlcNAc2) is transferred to nitrogen in the side chain of some Asn residues

that are part of the sequon. This process involves a ternary interaction of the glyco-

sylation sequon with an oligosaccharyl transferase and the dolichol phosphatelinked

Contributed by Louise Royle, Raymond A. Dwek, and Pauline M. RuddCurrent Protocols in Protein Science (2006) 12.6.1-12.6.45

Copyright C 2006 by John Wiley & Sons, Inc.

Glycosylation

12.6.1

Supplement 43


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OligosaccharidesN- and O-Linkedto Glycoproteins

12.6.2

Supplement 43 Current Protocols in Protein Science

oligosaccharide precursor, both of which are located in the membrane of the ER. Each

sugar chain is subsequently processed in the ER and Golgi by a series of glycosidases

and glycosyltransferases to either an oligomannose or a hybrid- or complex-type gly-

can. In mammals, oligomannose sugars contain five to nine mannose residues attached

to the GlcNAcGlcNAc (chitobiose) core. Hybrid glycans are those in which only the

Man1,3Man1,4GlcNAc- arm of the core contains GlcNAc (Fig. 12.6.5). The addi-

tion of this residue by GlcNAc transferase I is the starting point for the synthesis of both

hybrid and complex glycans. In the case of complex glycans, GlcNAc transferase II adds

a GlcNAc residue to the Man1,6Man1,4GlcNAc- arm of the core, allowing future

processing to bi-, tri-, and tetraantennary complex glycans.

The practical process of assigning monosaccharide, linkage type, and position to glycans

in oligosaccharide pools, which is the subject of this unit, is made considerably easier if

a symbolic, rather than the classical IUPAC nomenclature, is adopted to represent glycan

structures. The scheme here uses shapes to represent monosaccharides, various fills to

represent modifications to the monosaccharide, bond angle to represent linkage position,

and line type to represent anomericity. This symbolic representation is designed for ease

of use, and does not represent the true conformation of the glycans. The nomenclature

is explained in Figures 12.6.1 and 12.6.2, and the structures of N-glycan core structures,

hybrid, complex, and oligomannose-type N-linked oligosaccharides, and the O-glycan

core structures are shown in Figures 12.6.1 to 12.6.8.

Glycans are frequently as large as the protein domains to which they are attached.

Moreover, in addition to stabilizing the protein (Joao et al., 1992), both the sugars

themselves and the N-glycosidic linkage to the Asn side chain are flexible. This feature

enables the glycans to shield large areas of the protein surface (Woods, 1995). As

mentioned above, it is clear that a detailed understanding of the roles of glycosylation

can only be achieved when the protein and sugar(s) are viewed as an entity. By combining

glycan sequencing data and oligosaccharide structural information (Petrescu et al., 1999)

with protein structural data, many glycoproteins can now be modeled in their entirety.

This means that, in many cases, it is possible to visualize the sugars in the context of the

glycoproteins to which they are attached. An example of this is shown in Figure 12.6.9, in

which the N-linked, O-linked, and GPI anchors of CD59 and CD55 have been modeled

by combining protein and glycan structural data with the oligosaccharide database.

Rapid, sensitive, state-of-the-art oligosaccharide sequencing technology is needed to

deal with the complex heterogeneity of the glycoform populations of many natural

glycoproteins. Figure 12.6.10 highlights the strategies that have been developed at the

Glycobiology Institute that enable the overall N- and O-glycans released from 5 to 30 g

of glycoprotein to be analyzed in a few days using HPLC (see Basic Protocol 6; Guile

et al., 1994, 1996; and Royle et al., 2002) and/or MS (see Chapter 16 and UNIT 12.7;

Rudd et al., 1997a) and applied to biologically important molecules (Royle et al., 2003;

Rudd et al., 2002; Tekoah et al., 2004). N-linked glycans can be released directly from

SDS-PAGE gels (UNIT 10.2; Kuster et al., 1997), or the N- and O-glycans can be released

from purified glycoproteins by hydrazine (see Basic Protocol 4). These advances open

up the possibility of ranking high-throughput sequencing of oligosaccharides alongsidethat of proteins and DNA.

This unit deals with the practical aspects of a state-of-the-art strategy for analyzing the

structure of glycosyl side-chains on glycoproteins (for an overview of glycosylation

analysis, see UNIT 12.1). This involves four stages: (1) glycan release, (2) labeling the

glycan pool, (3) separating (profiling) the glycans in the pool, and (4) characterization.

Each of these steps is discussed in the sections that follow.


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Glycosylation

12.6.3

Current Protocols in Protein Science Supplement 44

Figure 12.6.1 Various principles of diagrammatic nomenclature. (1) Each monosaccharide isrepresented by a particular symbol (e.g., square for glucose) whose shape does not relate to

structure. (2) The various fillings of the shape represent substituents on the monosaccharide

(e.g., open, not substituted; filled in, N-acetyl group; dot, deoxy sugar). Hence, galactose is

represented by an open diamond, N-acetyl galactosamine by a filled diamond, and deoxygalactose

(fucose) by an open diamond with a dot inside. When the exact monosaccharide is not known

(e.g., from MS data), an appropriately filled hexagon is used (e.g., open hexagon, hexose; filled

hexagon, HexNAc). (3) Linkage positions are represented by the angle of the line linking adjacent

monosaccharides. As most sugars are linked via the C1 reducing end (except sialic acids, which

are linked via C2), this information is not represented in these diagrams. It is the position to which

the reducing end of the sugar to the left attaches to the sugar on the right that is represented

by the bond angle. (4) The type of line represents anomerity. A straight, solid line indicates a

beta linkage and a dotted line represents an alpha linkage. Unknown linkages are represented

by a wavy line. (5) Substituents can be represented by letters (e.g., S for sulfate). This simple

visual representation conveys all the exact known or unknown structural information needed when

working with complex glycans without the need for colors.

RELEASE OF GLYCANS FROM GLYCOPROTEINS

Intact N-linked glycans can be released from glycoproteins or glycopeptides enzymat-

ically with peptide N-glycosidase F (PNGase F; Roche Diagnostics), as described in

Basic Protocol 1. This enzyme is an amidase that cleaves the linkage between the core

GlcNAc and the Asn residue of all classes of N-glycans (Fig. 12.6.3; Kuhn et al., 1994),

with the exception of some plant and insect N-glycans that contain fucose 1,3-linked

to the core GlcNAc residue attached to the protein. The glycoprotein should be fully

denatured, since unfolding promotes enzyme accessibility to the cleavage site, which

may, in some cases (as with RNase B), be protected by the native protein (Fig. 12.6.11).

In some cases it may also be necessary to cleave the peptide chain with trypsin before

PNGase F treatment to ensure that all sugars are released. Although initially released as

the glycosylamine, the amino group at the reducing terminus rapidly converts to hydroxyl

or can be encouraged to do so at low pH. Thus, NaHCO 3, not NH4HCO3, should be

used as a buffer for glycan release, since the latter promotes retention of the amine group

at the reducing terminus of the glycans, resulting in severe losses during 2-AB labeling

(Kuster et al., 1997).


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12.6.4


Figure 12.6.2 Examples showing how glycan structures are constructed.

Figure 12.6.3 Native N-linked oligosaccharide core structures. For nomenclature, see Table12.6.8.

PNGase F can be used to release sugars from glycoproteins that are either in solution

(see Basic Protocol 1) or in an SDS-PAGE gel band (see Basic Protocols 2 and 3).

One advantage of working from SDS-PAGE gels is that the electrophoresis step is a

useful means of purifying many glycoproteins to homogeneity without prohibitive loss

of material. This means that it is possible to analyze the glycosylation of low levels of

biologically relevant glycoproteins where either limited quantities of material or protein

purification problems would previously have precluded a detailed glycan analysis. In

practice, releasing the sugars directly from a glycoprotein in a gel band results in a higher

recovery of glycans than that obtained when the glycoprotein is incubated with PNGase F

in solution. A third application for the in-gel method is to release the sugars from peptide

fragments resulting from protease digestions when these can be resolved by SDS-PAGE.

In some cases, this may enable a rapid analysis of glycosylation sites in glycoproteins.

Reducing gels also allow the straightforward separation of proteins into their component

subunits. For example, IgG can be resolved into heavy and light chains (K uster et al.,

1997), and the surface coat proteins of the hepatitis B virus or particle can be separated

into three major glycoprotein components (Mehta et al., 1997). Also, when gelatinase

B is purified on gelatin-Sepharose, it is contaminated with its natural inhibitor (tissue

inhibitor of metalloproteinase I or TIMP-1). This enzyme-inhibitor complex is stable,

but SDS-PAGE run under reducing conditions can be used to resolve the two proteins.


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Glycosylation

12.6.5


Figure 12.6.4 Oligomannose-type oligosaccharides. D1, D2, and D3 refer to the terminal mannose residuesattached 1,2 to the Man1,6Man1,6 (D1), the Man1,3Man1,6 (D2), and the Man1,2Man1,3 (D3)

arms of the glycan core.

N-linked glycans can then be released directly from the gels with PNGase F (Rudd et al.,

1999a).

A final advantage of the in-gel release method is that the protein remains in the gelafter the sugars have been removed. In-gel proteolysis of the protein (for example, by

trypsin) and analysis of the peptide fragments by nanospray MS enables the protein to

be identified. This is achieved by using a protein data base to compare the molecular

weights of the tryptic fragments with those of the predicted sequences of fragments from

tryptic digests of known proteins (Kuster et al., 1997).

While PNGase F is the enzyme of choice to release N-linked sugars for glycan anal-

ysis, in certain circumstances other enzymes may be required (see UNIT 12.4 for more

details). For example, PNGase A cleaves sugars containing 1,3-linked core fucose,


6/45


12.6.6


Figure 12.6.5 Hybrid and complex 2-AB-labeling oligosaccharides. For nomenclature, see Table12.6.8.

which are not susceptible to PNGase F. Two enzymes that cleave between the two

N-acetylglucosamine (GlcNAc) residues within the chitobiose core are endoglycosidase

D (endo D), which releases all classes of N-linked sugars, and endo H, which selectively

cleaves oligomannose- and hybrid-type structures. Another useful group of enzymes is

the endo--N-acetylglucosaminidases F1, F2, and F3. All three enzymes cleave within

the chitobiose core, but while endo F1digests only oligomannose and hybrid structures,

endo F2 cleaves oligomannose and biantennary glycans, and endo F3 is specific for bi-and triantennary sugars.

Both N- and O-linked sugars can be released chemically with hydrazine (Fig. 12.6.12)

(see Basic Protocol 4). O-linked glycans may only be released chemically, because a

generic O-glycanase is not yet available.


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Glycosylation

12.6.7


Figure 12.6.6 Sialylated complex 2-AB-labeled N-linked oligosaccharides. For nomenclature,

see Table 12.6.8.

Figure 12.6.7 O-glycan core structures.

Figure 12.6.8 Molecular models of O-glycan core structures.


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12.6.8


Figure 12.6.9 (A) CD55 (also known as decay accelerating factor or DAF) and ( B) CD59 both containN- and O-linked glycans and are attached to the membrane by a glycosylphosphatidylinositol (GPI)

anchor. CD59 is a glycoprotein consisting of one domain (Ly6) and it is located close to the membrane

surface of many cells, including erythrocytes. Both the classical and alternative complement pathways

terminate with the formation of the membrane attack complex (MAC) in the cell surfaces of bacteria

and other pathogens, and this leads to cell lysis. Host cells are normally protected from destruction

through inhibitors of the complement pathway, such as CD55, which destabilizes the C3 convertase

components C3b/Bb and C4b2a, and CD59, which binds C8 and/or C9 preventing the formation of the

fully assembled MAC complex. CD59 contains all three types of post-translational modifications that

involve glycosylation. It has one N-linked sugar at Asn-18 and one or two O-linked glycans (Rudd et

al., 1997b). CD55 is also linked to the erythrocyte cell surface by means of a GPI anchor. The protein

consists of four complement control protein domains. Three of these domains, together with an O-linked

region that acts as a rigid spacer, act to position the N-linked glycan some 20 nm above the membrane

surface (Kuttner-Kondo et al., 1996).

LABELING THE RELEASED GLYCANS

The classical method of attaching the radiolabel tritium to the released oligosaccharides

has been replaced by labeling with fluorescent compounds. As a result, it is now possible

to detect sugars in the femtomole range instead of the picomole range. A suitable label

must have a high molar labeling efficiency and be capable of labeling the oligosaccharide

components of a glycan pool nonselectively so that they are detected in their correct

molar proportions. As described in Basic Protocol 5, 2-aminobenzamide (2-AB) fulfills

these conditions and, in addition, it is compatible with a range of separation techniques


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Glycosylation

12.6.9


Figure 12.6.10 Flowchart for analysis of N-glycans.

including normal-phase (NP), weak anion-exchange (WAX), and reversed-phase (RP)

HPLC, matrix-assisted laser desorption/ionization MS (MALDI-MS), and electrospray

ionization MS (ESI-MS). It was for these reasons that 2-AB was selected as the label for

the HPLC-based glycan analysis strategy described below.

PROFILING BY HPLC

Three different HPLC systems, NP-HPLC, RP-HPLC, and WAX-HPLC (see Basic Proto-

col 6) are used to assign structures to the released sugars. If material is limited, NP-HPLC

should be used, as it gives the maximum information from the minimum sample. Al-

though it is possible to recover the sample from a MALDI-MS target, in practice, roughly

twenty NP- or RP-HPLC or five WAX-HPLC runs can be carried out with the amount

of sample needed for one MALDI-MS analysis. Ten to fifty femtomoles of a single gly-

can give a reasonable signal-to-noise ratio on NP- or RP-HPLC. The amount of sample

needed from a pool depends on the number of different glycans in the pool and on its

concentration. The best way to determine how much to load is to run a small amount of

the sample (e.g., 1%) and adjust the volume for subsequent runs depending on the ratioof signal to noise in the trial run.

A major advantage of all HPLC systems is that they can be used preparatively. This

means, for example, that pools of sugars separated according to charge using WAX-

HPLC can be collected and individually analyzed by NP-HPLC. This procedure may

be used to limit the number of structures in the normal-phase profiles, thus simplifying

the interpretation. Preparative HPLC also allows individual peaks to be collected and

analyzed by mass spectrometry (UNIT 12.7) or other techniques to validate assignments

(Rudd et al., 1997a).


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12.6.10


Figure 12.6.11 Structures of CD59, RNase B, and PNGase F. The structure of PNGase F isbased on the X-ray crystal structure (Norris et al., 1994). The protein is folded into two domains,

each with an eight-stranded antiparallel jelly roll configuration similar to the types of structures

found in lectins. CD59 was modeled according to the NMR solution structure (Kieffer et al., 1994).

RNase B was modeled according to the X-ray crystal structure (Williams et al., 1987). All threemolecules are modeled to the same scale. The active site for PNGase F is in a cleft flanked by five

Trp residues and one Phe residue (Kuhn et al., 1994). In contrast to the CD59 Asp-sugar amide

linkage, which is susceptible to the enzyme, the same linkage in RNase B is protected by the

protein and is not accessible. The arrow in CD59 indicates the attachment site for the GPI anchor

in the membrane-bound form.

WAX-HPLC

WAX chromatography using a column such as GlycoSep C (Prozyme) is used to separate

glycan mixtures according to the number of charged residues they contain (Guile et al.,

1994). Within one charge band several peaks may be resolved, since larger structures

elute before smaller ones. Charge due to sialic acid can be assigned by reference to astandard run of fetuin oligosaccharides, which contain mono-, di-, tri,- and tetrasialylated

glycans (Fig. 12.6.13A). Neutral sugars (and noncarbohydrate material) elute in the void.

Incubation of the N-glycans from prostate-specific antigen with a sialidase specific for

2-3 linked sialic acids shows that these glycans possess a mixture of both 2-3 and 2-6

sialic acids (Fig. 12.6.13B,12.6.13C), whereas incubation withArthrobacter ureafaciens

sialidase digests all of the sialylated glycans to neutral.


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Glycosylation

12.6.11


Figure 12.6.12 The chemistry of thehydrazine reaction to release intact O-glycans from proteins,the 2-AB fluorescent labeling step, and the peeling reaction. The exact mechanism of release of

intact O-glycans has not been determined but is believed to take place via an initial base-catalyzed

-elimination reaction cleaving the bond between GalNAc and Ser/Thr, followed by immediate

reaction of the released aldehyde with excess hydrazine to form a hydrazone derivative. In the

presence of excess water, further -elimination may take place when the GalNAc is substituted at

the 3 position (as shown by the Gal R1 group in this figure), which leads to loss of the GalNAc and

may proceed even further to total destruction of the glycan. The hydrazone is then re-N-acetylated,

the acetohydrazone at C1 is cleaved, and a glycan with a free reducing terminus is produced. This

may then be reacted with 2-aminobenzamide (2-AB) to give an unstable Schiffs base intermediate,

which is stabilized by addition of sodium to the reaction mixture to form a reduced, fluorescently

labeled product of the glycan.

NP-HPLC

This is performed using an amide column in which the polar functional groups interact

with the hydroxyl groups on the sugars (Guile et al., 1996). Samples applied in high

concentrations of organic solvent adsorb to the column surface and are eluted with an

aqueous gradient so that glycans are resolved on the basis of hydrophilicity. Typically,

larger oligosaccharides are more hydrophilic than smaller ones, and require higher con-

centrations of aqueous solvent to elute. The system described here is sensitive and highly

reproducible, and has been developed using the GlycoSep N column (Prozyme). It is


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12.6.12


Figure 12.6.13 WAX separation showing that the N-glycans from prostate specific antigen (PSA)contain a mixture of both 2-3 and 2-6 sialic acids. (A) A standard mixture of mono-, di-, tri-, andtetrasialylated fetuin N-glycans used for calibrating the column. (B) N-glycans from low-pI PSA untreatedand (C) N-glycans from low pI PSA treated with NANI, which digests terminal 2-3 sialic acids.

Figure 12.6.14 NP-HPLC separation of the N-glycans released from low-pI prostate-specificantigen. The gel from which the Coomassie blue-stained band of low pI PSA was excised is also

depicted.


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Glycosylation

12.6.13


capable of resolving subpicomolar quantities of mixtures of fluorescently labeled neutral

and acidic N- and O-glycans simultaneously and in their correct molar proportions. A

typical profile is shown in Figure 12.6.14. Elution positions are expressed as glucose

units (GU) by comparison with the elution times of a dextran ladder. The contribution

of individual monosaccharides to the overall GU value of a given glycan can be calcu-

lated, and these incremental values can be used to predict the structure of an unknown

sugar from its GU value (Tables 12.6.1 and 12.6.2). The system is able to resolve arm-

specific substitutions of a multiantennary sugar, and the linkage positions of individual

Table 12.6.1 N-Linked Oligosaccharides

Structurea Massb GUc AUc

Core structures

N2M1 586.32 2.65 2.72

F(6)N2M1 732.38 3.17 4.95

Man3 910.44 4.41 2.56

F(6)Man3 1056.5 4.90 4.65

Oligomannose structures

Man9 1882.80 9.56 0.83

Man8 1720.74 8.83 0.83

Man7 1558.68 7.96 1.3

Man6 1396.62 7.10 1.52

Man5 1234.56 6.21 2.17

Bi-, tri-, and tetraantennary complex structures

A2 1316.59 5.50 3.14

A2B 1519.67 5.63 3.10

F(6)A2 1462.64 5.93 5.18

A2G2 1640.50 7.15 3.22

A2BG2 1843.60 7.30 4.96

F(6)A2G2 1786.60 7.57 5.18

F(6)A2BG2 1989.7 7.67 7.59

A3 1519.6 5.91 3.65

A3G3 2005.6 8.32 3.81

A4 1722.70 6.53 2.70

A4G4 2371.00 9.67 2.70

Sialylated structures

A2G2S(3)1 1931.69 7.49 3.84

A2G2S(6)1 1931.69 7.89 3.48F(6)A2G2S(3)1 2077.75 8.14 5.86

F(6)A3G3S(6,6)2 3845.37 9.61 4.46

F(6)A3G3S(6,6,3)3 4136.46 9.54 9.78

aN-linked structures are selected as examples only; this is not an exhaustive list. All of these structures

are illustrated schematically in Figures 12.6.3 to 12.6.6. For nomenclature, see Table 12.6.8.bMass given is for closed-ring unlabeled structures; add 120.069 for addition of 2-AB label and 22.9 for

sodium adduct in MALDI-MS.cGlucose unit (GU) and arabinose unit (AU) values are given for 2-AB-labeled structures.


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12.6.14


Table 12.6.2 Incremental Glucose Units for Addition of Monosaccharides to N-LinkedOligosaccharides

Monosaccharide Linkage To Increment

Core fucose 1,6 Chitobiose and Man3 0.5

Core fucose 1,6 A2, A2G2, A2G2B,

A3G3

0.42

Core fucose 1,3 Any structure 0.8

Antennary fucose 1,3; 1,6 Any structure 0.40-0.80a

Mannose 1,2; 1,3; 1,6 Man3 up to Man7 0.9

Mannose 1,2; 1,3; 1,6 Man8 and above 0.72

Bisecting GlcNAc 1,4 First mannose 0.10-0.15

Galactose 1,4 Any structure 0.90-1.00

NeuNAc 2,3 Galactose of any

structure

0.7

NeuNAc 2,6 Galactose of any

structure

1.15

aDepending on location.

monosaccharides, such as fucose, also affect retention times, giving a further level of

specificity.

RP-HPLC

Reversed-phase HPLC, using a GlycoSep R C18 column (Prozyme), separates sugars

on the basis of hydrophobicity. Samples are applied in an aqueous solvent and eluted

with increasing concentrations of organic solvent. The column is calibrated with an

arabinose ladder rather than the dextran ladder used for the NP-HPLC, and retention

measurements are made in arabinose units (AU; Guile et al., 1998). Sugars that co-

elute by NP-HPLC can often be separated using RP-HPLC (Fig. 12.6.15). The effect

of linkage position is much more marked on reversed-phase separations than on normal

phase. For example, the two trisaccharides Lewis-X and Lewis-A, which have the same

monosaccharide compositionGal1,4Fuc1,3GlcNAc and Gal1,3Fuc1,4GlcNAc,

respectivelyhave similar elution times on normal phase (Lex GU value: 2.59; Lea GU

value: 2.85), but are well separated by reversed phase (Lex AU value: 0.00; Lea AU

value: 2.39). RP-HPLC is particularly useful for identifying oligosaccharides containing

bisecting GlcNAc. Structures that differ only by the presence or absence of bisecting

GlcNAc are well separated on reversed-phase columns, but elute at similar positions on

normal-phase columns.

CHARACTERIZATION OF N- AND O-LINKED OLIGOSACCHARIDESPreliminary Assignment of Peaks from NP- and RP-HPLC Profiles

Tables 12.6.1 and 12.6.2 and Figure 12.6.16 show the GU and AU values for common N-

and O-linked glycans. They are used to list the possible structures that correspond to the

GU (NP-HPLC analysis) and AU (RP-HPLC analysis) values of the sample peaks. These

values are highly reproducible from column to column and system to system (Guile et al.,

1996; Royle et al., 2002), giving typical standard deviations of


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Glycosylation

12.6.15


Figure 12.6.15 Two-dimensional separation of O-glycans using NP- and RP-HPLC. GU and AUvalues for a set of digests carried out on an O-linked tetrasaccharide and monitored by NP- and

RP-HPLC. On the NP column, the peak that results from digesting the glycan with SPG (B; GU2.73) elutes at a smaller GU than the starting glycan (A; GU 3.58); while on the RP column, thestarting glycan and the product of the digest elute in the same position. However, comparing the

BTG digest to the starting glycan (DandA), the GU of the product (1.79) is again less than the GUof the starting material (3.58) on the NP column, whereas the AU of the product (2.80) is greater

than the AU of the starting glycan (1.63) on the RP column. Thus, the two separation techniques

are complementary, allowing two structures that co-elute on one column to be resolved on the

other. Abbreviations: BTG, bovine testes -galactosidase; SPG, Streptococcus pneumoniae-

galactosidase; SPH,Streptococcus pneumoniae-hexosaminidase; undig, undigested.

Oligosaccharide Structural Analysis Using Exoglycosidase Sequencing

Enzymatic analysis of oligosaccharides using highly specific exoglycosidases is a power-

ful means of determining the sequence and structure of glycan chains (see Basic Protocols

7 and 8). The preliminary assignments of structures made from GU and AU values are

confirmed by digesting the entire glycan pool with arrays of exoglycosidases, and the di-

gestion products are analyzed by NP-HPLC using the same conditions as for the original

glycan pool. However, until recently, this has only been possible after isolation of single


16/45


12.6.16


Figure 12.6.16 Structure and mass of O-linked oligosaccharides. The structures are selectedas examples only and do not represent an exhaustive list. Mass given is for closed-ring unlabeled

structures; add 120.069 for addition of 2-AB label, and 22.9 for sodium adduct in MALDI-MS. GU

and AU values are given for 2-AB-labeled structures.


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Glycosylation

12.6.17


sugars from the total glycan pool and digesting each with exoglycosidase enzymes, either

sequentially or using enzyme arrays. It is often impractical and always time consuming to

purify each individual sugar. Therefore, the simultaneous analysis of the total glycan pool

as presented in Basic Protocol 7 (for N-glycans) and Basic Protocol 8 (for O-glycans) is

a major development.

Detection of Structures by Mass Spectrometry

Mass spectrometry is used mainly to determine the molecular weights and hence the

composition of the glycans in the pool, but does not give linkage information directly.UNIT 12.7presents methods to analyze glycan structure by this method.

CAUTION: Acrylamide is a neurotoxin, although it is not as dangerous once it has

been polymerized.N,N,N,N-Tetramethylethylenediamine (TEMED) is also toxic. Wear

gloves at all times and use these compounds in a fume hood. Refer to materials safety

data sheets (MSDSs) for further information.

BASIC

PROTOCOL 1

ENZYMATIC RELEASE OF N-LINKED OLIGOSACCHARIDES BY PNGase FDIGESTION IN SOLUTION

This enzymatic procedure (Goodarzi and Turner, 1998) may be used to release N-

linked glycans from glycoproteins when the glycoprotein sample is pure and when use ofhydrazinolysis (Basic Protocol 4) is not feasible. As there may be variable steric hindrance

depending on the nature of the peptide and/or the glycans, the glycoprotein is denatured

by reduction and alkylation in the presence of SDS. The SDS must then be exchanged

for another detergent, since SDS inactivates PNGase F. Finally, since it is possible that

glycan removal may be incomplete, it is advisable to check for the completion of release

by SDS-PAGE. Incomplete removal is indicated by the presence of more than one band.

Materials

Glycoprotein sample

Buffer A (see recipe)

Buffer B (see recipe)10% (w/v) dithiothreitol (DTT)

1000 U/ml peptide N-glycosidase F (PNGase F), recombinant glycerol-freelyophilate (Roche Life Sciences)

Toluene

Sub-boiling-point-distilled (SBPD) water

Vacuum centrifuge

100C temperature-controlled heating block

Protein-binding membrane (e.g., Micropure-EZ centrifugal filter devices,Millipore.)

PCR tubes

Additional reagents and equipment for SDS-PAGE (UNIT 10.1)

1. Dry a glycoprotein sample in a 1.5-ml microcentrifuge tube in a vacuum centrifuge.

Generally 50 to 200g of glycoprotein is required. The glycoprotein should be isolatedaccording to usual procedures. If the sample is in a low-molarity or volatile salt solution,

no dialysis is required.

The incubation of a control glycoprotein with known glycosylation alongside experimentalsamples is recommended. Suitable control glycoproteins include ribonuclease B, bovine

serum fetuin, and haptoglobin. All are available from Sigma, although there may be somevariation in glycosylation profiles between different batches.


18/45


12.6.18


2. Dissolve sample in 10 l buffer A and incubate 3 min at 100C.

3. Cool to room temperature and add the following in the order indicated:

8 l buffer B

2 l 10% DTT

10 l 1000 U/ml PNGase F (10 U)

5 l toluene.

Toluene is added to prevent bacterial growth.

4. Incubate 36 hr at 37C.

5. Remove 5 l and analyze by SDS-PAGE (UNIT 10.1). When the sample is completely

deglycosylated, proceed with step 7. Otherwise continue incubation.

If the reaction mixture is analyzed alongside a control sample that has not been incubated

with PNGase F, a shift to a lower molecular weight, corresponding to the deglycosy-lated protein, will be observed. A series of bands may be observed that can indicate

deglycosylation at different sites of the glycoprotein.

6. Wash the Micropure-EZ centrifugal filter devices by adding 200 l SBPD water and

microcentrifuging 10 min at 7000 g, room temperature. Discard washings.

7. Apply the digested glycan sample to the filter (in the middle) and microcentrifugethe filter 2 min at maximum speed, room temperature, keeping any eluate.

8. Wash glycan sample off the filter with 100 l SBPD water, microcentrifuge 5 min at

1400 g, room temperature, and pool the eluate with that from step 7.

9. Dry eluate in a vacuum centrifuge.

The sample is ready for 2-AB labeling or may be stored frozen (up to 1 year at 20C)after redissolving in 20l SBPD water.

BASIC

PROTOCOL 2

IN-GEL ENZYMATIC RELEASE OF N-LINKED GLYCANS BY PNGase FFROM PROTEINS SEPARATED IN SDS-PAGE GEL BANDS

The following in-gel release method has been developed to release N-glycans fromCoomassie bluestained protein bands on SDS-PAGE gels by PNGase F (Kuster et al.,

1997). The released glycan pool can be analyzed by matrix-assisted laser desorption/

ionization time-of-flight MS (MALDI-TOF-MS) and, after fluorescent labeling, by

HPLC. The individual sugars in the pool can be analyzed simultaneously using enzyme

arrays (Guile et al., 1996) monitored by either MALDI-TOF-MS or HPLC.

Materials

30% acrylamide/0.8% bis-acrylamide solution (e.g., Protogel; NationalDiagnostics)

0.5 M Tris base, pH 8.8

1.5 M Tris base, pH 6.6

Sub-boiling-point-distilled (SBPD) water10% (w/v) SDS

10% (w/v) ammonium persulfate (APS)

N,N,N,N-Tetramethylethylenediamine (TEMED)

Glycoprotein sample

0.5 M dithiothreitol (DTT)

5 SDS sample buffer (see recipe)

100 mM iodoacetamide

25 mM Tris/190 mM glycine/0.5% (w/v) SDS


19/45

Glycosylation

12.6.19


50% (v/v) methanol/7% (v/v) acetic acid

5% (v/v) methanol/7% (v/v) acetic acid

20 mM NaHCO3, pH 7.0

1:1 (v/v) acetonitrile/20 mM NaHCO31000 U/ml peptide N-glycosidase F (PNGase F) in 20 mM NaHCO3Acetonitrile

Dowex AG50X12 (H+ form; 100 to 200 mesh; Bio-Rad), activated (see UNIT12.7forconditioned resin)

1 M HCl

70C water bath or heating blockRoller-mill-type mixer (Spiramix from Denley Instruments, or equivalent)

Vacuum centrifuge

Sonicator

0.45-m Millex-LH/hydrophilic PTFE filter (Millipore)

2.5-ml syringe, lubricant-free

Additional reagents and equipment for SDS-PAGE (UNIT 10.1) and Coomassie bluestaining (UNIT 10.5)

NOTE:Iodoacetamide is light sensitive. It should be prepared fresh before each use.

Run gel

1. Prepare an 80 80 1mm polyacrylamide gel as described in UNIT 10.1(Laemmligel method), but use the gel recipes given in Table 12.6.3. Always add APS and

TEMED immediately before pouring gels.

The 10% APS solution must be prepared fresh every time. If it fizzes, it is still active.

The greater the percentage of bis-acrylamide the greater the cross-linking of the gel and

hence the smaller the pore size. Higher bisacrylamide concentrations lead to reducedglycan yield. Gels should be 15% for 10 to 70 kDa proteins, 10% for 70 to 200 kDa

proteins, or 6% for200 kDa proteins.

Reduce and alkylate protein

2. To 5 to 30 g glycoprotein, add 0.5 M DTT to a final concentration of 50 mM and

5 SDS sample buffer to a final concentration of 1. Incubate 10 min at 70C.

Reduction and alkylation are necessary to ensure optimal efficiency in digests using

PNGase F and/or trypsin.

Table 12.6.3 Preparation of Separating and Stacking Gels

Separating gels Stacking gel

6% 10% 15% 4%

30% acrylamide/0.8%

bis-acrylamide

2.4 ml 4.0 ml 6.0 ml 1.33 ml

0.5 M Tris base, pH 8.8 3.0 ml 3.0 ml 3.0 ml

1.5 M Tris base, pH 6.2 2.5 ml

Distilled water 6.6 ml 5.0 ml 3.0 ml 6.1 ml

10% SDS 120 l 120 l 120 l 100 l

10% APS 120 l 120 l 120 l 50 l

TEMED 12 l 12 l 12 l 10 l

Total 12.252 ml 12.252 ml 12.252 ml 10.09 ml


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12.6.20


Clearly the amount of protein required to release picomoles of sugar for analysis willdepend on the molecular weight of the protein as well as how many glycosylation sites are

occupied. For example, in the case of ovalbumin, which has a mol. wt. of 45 kDa and oneglycosylation site, 4.5 g protein equals 100 pmol. Note that overloading gels does not

always yield more glycans. The protein bands should be as tight as possible. The methodworks better on a small scale; therefore, the gel pieces to be processed should be small

as well.

3. Add 100 mM iodoacetamide to a final concentration of 10 mM and incubate 30 min

at room temperature in the dark.

4. Apply sample to a gel slot in a vertical mini-gel system and run at 25 mA of constant

current per gel. Use 25 mM Tris/190 mM glycine/0.5% SDS as the running buffer.

Stain/destain protein

5. Visualize protein in the gel by staining with Coomassie blue (UNIT 10.5) for 2 hr.

6. Partially destain by rinsing with 50% methanol/7% acetic acid for a few minutes and

then with 5% methanol/7% acetic acid for a minimum of 2 hr at room temperature.

Use


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Glycosylation

12.6.21


16. Repeat the procedure described in step 15 twice with 200 l water and once with

200 l acetonitrile, then once with 200 l water and once with 200 l acetonitrile.

Add each wash to the supernatant.

The acetonitrile shrinks the gel.

17. If desalting is not required, go directly to the filtration in step 19. If desalting is

required for MALDI-MS, then add 30 l activated Dowex AG50X12 (H+) slurry to

the sample, vortex, and incubate 5 min at room temperature to desalt.

18. Centrifuge 5 min at 550 g, room temperature.19. Filter the supernatant with a 0.45-m Millex-LH/hydrophilic PTFE filter attached to

a 2.5-ml syringe, collecting the filtrate in a 1.5-ml microcentrifuge tube.

The sample can now be stored frozen at20C for up to a year. It can be cleaned up as

described later for direct analysis by MALDI-MS or can be labeled with 2-AB (see BasicProtocol 5).

BASIC

PROTOCOL 3

IDENTIFICATION/CONFIRMATION OF GLYCOPROTEIN BY IN-GELTRYPSIN DIGESTION

This procedure is based on the method of Kuster et al. (1997). After the sugars have been

released, the identity of the protein can be confirmed by in situ trypsin digestion, MS

analysis of the recovered fragments, and comparison of the obtained masses or peptidesequences with a protein database. Trypsin digests can be performed either directly on

the reduced and alkylated glycoprotein or after in situ deglycosylation with PNGase F.

Materials

Gel pieces containing glycoprotein of interest

Acetonitrile

20 and 50 mM ammonium hydrogen carbonate (NH4HCO3) insub-boiling-point-distilled (SBPD) water

0.1 mg/ml sequencing-grade trypsin (Roche Diagnostics) solution in SBPD water

10% (v/v) formic acid in SBPD water

Vacuum centrifuge

Wash gel pieces

1. Place gel pieces in 1.5-ml microcentrifuge tubes.

2. Wash 15 min in 500 l acetonitrile. Discard acetonitrile.

3. Wash 15 min in 500 l of 50 mM NH4HCO3. Discard NH4HCO3.

4. Repeat steps 2 and 3.

5. Dry gel pieces in a vacuum centrifuge.

Digest with trypsin

6. Dilute 0.1 mg/ml sequencing-grade trypsin solution 1/8 (v/v) in 20 mM NH4HCO3(final 12.5 g/ml).

7. Add 15 l of 12.5 g/ml sequencing-grade trypsin solution and 5 l of 20 mM

NH4HCO3to the gel pieces.

8. Incubate 15 min at room temperature.


22/45


12.6.22


Retrieve peptide

9. Remove solution from gel pieces and collect into 0.5-ml microcentrifuge tubes.

10. Add 10 l of 10% formic acid and 10 l acetonitrile to the gel pieces.


12. Remove solution from gel pieces and pool with previously collected solution in the

microcentrifuge tube.

13. Add 15 l acetonitrile to the gel pieces.


15. Remove solution from gel pieces and pool with previously collected solution in the

microcentrifuge tube.

16. Dry sample in a vacuum centrifuge.

The sample is now ready for sequence analysis by mass spectrometry (UNIT 12.7).

BASIC

PROTOCOL 4

MANUAL HYDRAZINOLYSIS TO RELEASE N- AND O-GLYCANS

This procedure involves cleavage of the N- or O-glycosidic linkage with anhydrous

hydrazine under conditions designed to optimize the release and minimize the degradationof sugars. This method for the release of O-linked glycans leaves the reducing end of

the glycan available for labeling with a fluorescent tag. In the case of O-linked sugars,

peeling (Fig. 12.6.12) is difficult to eliminate, but the conditions in this protocol

generally give


23/45

Glycosylation

12.6.23


Miniert valve (Pierce/Perbio Science)

Glass syringe with Teflon plunger and stainless-steel needle

Acid-washed long Pasteur pipets

Incubation equipment at 60 and 85C

Vacuum pump and vapor trap

Poly-Prep disposable chromatography columns (Bio-Rad)

Rotary evaporator with clean glass tubes

30-mm wide Whatman 1 Chr chromatography paper

Pinking shears

Chromatography tank: 60 cm tall, equipped with solvent troughs and lidWaxed paper

Lubricant-free 2.5-ml syringes

0.45-m pore size, 13-mm diameter hydrophilic PTFE Millex-LCR filter(Millipore)

Dialyze and freeze dry sample

1. Dialyze 10 g to 2 mg glycoprotein sample (in a volume of1 ml) in a flow

dialyzer against 0.1% trifluoroacetic acid 16 hr in a 4C cold room at a flow rate of

0.5 ml/min to remove all traces of salts or detergents.

It is preferable to use a flow dialyzer, as dialysis bags can introduce carbohydrate im-

purities. If using dialysis bags, use a flask with nitrogen sparge at 4C. If using TFA tosolubilize the protein, dialyze at 4C to avoid hydrolyzing sialic acid residues.

2. Transfer dialyzed glycoprotein into the bottom of a glass hydrazinolysis tube using

an acid-washed long Pasteur pipet.

Do not add more than 1 ml at a time for freezing/freeze drying, as the glass tube maycrack as the liquid expands on freezing. If the sample is in a larger volume, freeze dry

1 ml, then add the next 1-ml aliquot and freeze dry again.

3. Fit the Miniert valve in the open position. Place the glass tube inside a clean 50-ml

plastic tube (to catch sample in case glass breaks during freezing).

4. Freeze carefully in liquid nitrogen while rotating the tube.

Rotating the tube helps avoid expansion of the sample as it freezes, and thus breakage ofthe glass tube.

5. Lyophilize overnight.

6. Remove final traces of water by subjecting the sample to cryogenic drying for

2 days, then shut the Miniert valve.

Perform hydrazinolysis

Perform steps 7 to 9 under argon.

7. Add 100 l dry hydrazine, using a glass syringe, to each hydrazinolysis tube and

flame seal under argon at atmospheric pressure.8. Incubate 6 hr at 60C for O-link glycan release. For N-link release, ramp up the

temperature to 85C at 10C/min, then incubate 12 hr at 85C.

O-glycan release conditions may also release low levels of N-glycans. N-glycan releaseconditions will release O-glycans, but they will be partially degraded.

9. Remove hydrazine under vacuum and condense hydrazine vapor in a trap. Complete

hydrazine removal by repeated addition of anhydrous toluene (five additions of 10

drops each), followed by evaporation. Immediately place samples on ice.


24/45


25/45

Glycosylation

12.6.25


24. To elute the sugars apply 1.5 ml water to the paper in the syringe, leave for 5 min,

and push through the filter into a clean tube. Repeat three more times and evaporate

all the filtrate to dryness.

25. Reconstitute the sample in 25 l water and transfer to a microcentrifuge tube. Wash

the collection tube three times with 25 l water. Store samples in solution at 20C

until ready for analysis (stable for several months).

The sample can now be stored frozen at20C for up to one year. It can be purified asdescribed for direct analysis by MALDI-MS (UNIT 12.7) or can be labeled with 2-AB (see

Basic Protocol 5).

BASIC

PROTOCOL 5

FLUORESCENT LABELING OF THE GLYCAN POOL WITH 2-AB

Commercial kits are available for efficient (100%) nonselective 2-aminobenzamide

(2-AB) labeling of salt-free glycans or glycan pools. Sugars are labeled at their reducing

terminus (Fig. 12.6.12) for high-sensitivity fluorescent detection (10 to 50 fmol sugar

yields an acceptable signal-to-noise ratio) following WAX-, NP-, and RP-HPLC.

Materials

Glyko Signal 2-aminobenzamide (2-AB) labeling kit (Prozyme, GKK-404) orLudgerTag 2-AB labeling kit (LT-KAB-A2;http://www.ludger.com)

Glycoprotein sampleSub-boiling-point-distilled (SBPD) water

Acetonitrile

200-l PCR tubes

65C incubator or heating block

GlycoClean S cartridges (Prozyme) or LudgerClean S cartridges(http://www.ludger.com) or 3MM chromatography paper (Whatman) cut into10 3cm pieces

100-ml glass beaker

Lubricant-free 2.5-ml syringes

13-mm diameter, 0.45-m pore size hydrophilic PTFE Millex-LCR filters(Millipore)

Rotary evaporator and acid-washed tubesVacuum centrifuge

1. Mix the 2-AB labeling kit components as directed by the manufacturer.

Excess labeling mixture can be stored for use later; if frozen immediately and stored at

20C until just before use, it may be kept for3 months.

2. Completely dry down a glycoprotein sample into the bottom of a PCR tube. Add

5 l labeling solution per 50 nmol glycan. Incubate 2 to 3 hr at 65C according to

the kit instructions.

3. If using GlycoClean S or LudgerClean S cartridges to purify the labeled samples,

follow the manufacturers instructions, then proceed to step 8. If using paper chro-

matography perform steps 4 to 7.

4. Spot the sample onto the paper strip 1 cm from the bottom. Allow sample to dry.

Place the paper in a 100-ml glass beaker containing enough acetonitrile to wet the

paper, but not touching the sample spot. Leave for 1 to 1.5 hr until any free label

has moved with the solvent front away from the sample spot (view with a UV light).

Allow to dry.

5. Cut out the sample spot and place in a 2.5-ml lubricant-free syringe fitted with a

13-mm, 0.45-m hydrophilic Millex-LCR filter.


26/45


12.6.26


6. Add 0.5 ml SBPD water to the syringe, leave 10 min, then filter and collect the eluant

in either an acid-washed glass tube for drying in a rotary evaporator or in a clean

1.5-ml microcentrifuge tube for drying directly in a vacuum centrifuge.

7. Repeat step 6 four more times.

8. Dry sample down completely.

9. Redissolve sample in 200 l SBPD water and transfer to a microcentrifuge tube. Dry

in a vacuum centrifuge.

10. Redissolve in a suitable volume of SBPD water (e.g., 100 l for HPLC analysis).

For initial HPLC analysis, generally no more than 1 to 10% of the sample is required.Thus, reconstitution in volumes of 100 l to 1 ml are appropriate. The sample is stable in

solution for several weeks at 4C, but should be frozen for extended storage.

BASIC

PROTOCOL 6

MULTIDIMENSIONAL SEPARATION AND PROFILING OF 2-AB-LABELEDGLYCANS USING NP-, RP-, AND WAX-HPLC

Complementary information can be obtained by resolving a glycan pool on several

systems. Figure 12.6.17 shows such an analysis of the glycan pool released from prion

protein isolated from Syrian hamster brains (Rudd et al., 1999b). An HPLC system

capable of delivering a highly reproducible gradient and a sensitive fluorescence detector

are required. The Waters system is detailed below, but others are also appropriate. The

more recent Waters 2475 detector is about 20 times more sensitive for the detection of

2-AB labeled glycans compared to the older 474 detector.

Materials

Glycoprotein sample

80% (v/v) acetonitrile

HPLC standards (see recipe): dextrose for NP-HPLC, arabinose for RP-HPLC, andfetuin N-glycans for WAX-HPLC

HPLC solvents appropriate for method:Solvent A (see recipe; NP): 50 mM formate, adjusted to pH 4.4 with ammonia

Solvent B (NP): HPLC-grade acetonitrileSolvent C (see recipe; RP): 50 mM formate, adjusted to pH 5 with triethylamineSolvent D (RP): 50:50 (v/v) solvent C/acetonitrileSolvent E (see recipe; WAX): 500 mM formate, adjusted to pH 9 with ammoniaSolvent F (WAX): 10:90 (v/v) methanol/water

Waters Alliance 2695 Separations Module: combined autosampler, injector,degasser, and column heater

Waters 474, Waters 2475, or Jasco Fluorescence Detector (for 2-AB, excitation:max330 nm, band width 16 nm; emissionmax420 nm, band width 16 nm)

Pentium PC

Waters Bus SAT/IN interface for data collection for Jasco detectors

Waters Bus LAC/E interface, internal board for PC

Column:Normal phase: 4.6 250mm TSKgel amide-80 column (Anachem), GlycoSep N

column (Prozyme), or LudgerSep N1 amide column (http://www.ludger.com);column temperature 30C

Reversed phase: 4.6 150mm, 3-m, 130-

A pore-size Hypersil ODS C18 column(Phenomenex)orGlycoSep R column (Prozyme); column temperature 30C

WAX: Vydac protein WAX, 7.5 50mm column (P/N 301 VHP 575) GlycoSepC column (Prozyme), or Ludger Sep C2 anion exchange column(http://www.ludger.com); column temperature ambient


27/45

Glycosylation

12.6.27


Figure 12.6.17 Resolution of the N-glycans attached to prion protein from scrapie-infected ham-ster brain (PrPSc27-30) using NP-, RP-, and WAX-HPLC.

Prepare sample

1. Prepare samples in 80% acetonitrile for NP-HPLC or in 100% water for WAX-and

RP-HPLC. Store samples at 4C.

It is advisable to add water to the vial first, followed by the sample in aqueous solution,and then the acetonitrile, as this reduces the risk of precipitation.

Perform HPLC

2a. For NP-HPLC: Run the start-up method (to preserve the life of the column) followed

by the separation method with no injected sample (this helps reproducibility). Then

run the dextran standard followed by the samples.


28/45


12.6.28


Start-up method (SUM-NP), run time 30 min:

0 min 0 ml/min 20:80 (v/v) solvent A/solvent B

4 min 1 ml/min 20:80

8 min 1 ml/min 95:5


16 min 1 ml/min 20:80

25 min 1 ml/min 20:80

26 min 0.4 ml/min 20:80

60 min 0.4 ml/min 20:80

61 min 0 ml/min 20:80

Separating method (SM-NP), run time 180 min:

0 min 0.4 ml/min 20:80 (v/v) solvent A/solvent B

152 min 0.4 ml/min 58:42

155 min 0.4 ml/min 100:0

157 min 1 ml/min 100:0

162 min 1 ml/min 100:0

163 min 1 ml/min 20:80

177 min 1 ml/min 20:80

178 min 0.4 ml/min 20:80

260 min 0 ml/min 20:80

2b. For RP-HPLC:Run the start-up method (to preserve the life of the column) followed

by the separation method with no injected sample (this helps reproducibility). Then

run the arabinose standard followed by the samples.

Start-up method (SUM-RP), run time 30 min:

0 min 0.05 ml/min 5:95 (v/v) solvent C/solvent D

5 min 1 ml/min 5:95





29 min 0.5 ml/min 95:590 min 0.5 ml/min 95:5


Separating method (SM-RP), run time 180 min:

0 min 0.5 ml/min 95:5 (v/v) solvent C/solvent D

30 min 0.5 ml/min 95:5

160 min 0.5 ml/min 85:15

165 min 0.5 ml/min 76:24

166 min 1.5 ml/min 5:95

172 min 1.5 ml/min 5:95

173 min 1.5 ml/min 95:5

178 min 1.5 ml/min 95:5179 min 0.5 ml/min 95:5

220 min 0.5 ml/min 95:5

221 min 0 ml/min 95:5

If the RP system has been unused for a while, the system should be purged with buffersbecause the concentration of acetonitrile in the system decreases with time.

Dextran hydrolysate is not suitable as a standard for the RP column, as it elutes in tooshort a time scale.


29/45

Glycosylation

12.6.29


Solvent D is designed to assist the reproducibility of the shallow gradient.

The last two lines of SM-RP enable the system to be shut down automatically.

2c. For WAX-HPLC (charge separation): Run the start-up method (to preserve the life of

the column) followed by the separation method with no injected sample (this helps

reproducibility). Then check the system by running a standard set of charged glycans

released from fetuin before loading samples.

Start-up method (SUM-WAX), run time 30 min:

0 min 0 ml/min 0:0 (v/v) solvent E/solvent F


90 min 1 ml/min 0:100

91 min 0 ml/min 0:100

Separating method (SM-WAX), run time 80 min:

0 min 1 ml/min 0:100 (v/v) solvent E/solvent F


25 min 1 ml/min 21:79

50 min 1 ml/min 80:20

55 min 1 ml/min 100:0

65 min 1 ml/min 100:0

66 min 2 ml/min 0:100

77 min 2 ml/min 0:100

78 min 1 ml/min 0:100

120 min 1 ml/min 0:100

121 min 0 ml/min 0:100

SUPPORT

PROTOCOL

CALIBRATION OF THE HPLC SYSTEM

It is essential to calibrate the normal- and reversed-phase HPLC systems against tem-

poral or other variations in factors such as column condition, pressure gradient, eluent

concentration, or environmental conditions. A least-squares fit fifth-order polynomial

curve-fitting calibration method is used for this purpose, employing a range of standard

peaks. This curve fitting may be performed either manually or by the use of computersoftware such as Waters GPC for Empower/Millennium chromatography management

systems.

An external standard (dextran ladder for NP or arabinose ladder for RP) is run indepen-

dently of the sample but under the same conditions. The elapsed time between the sample

run and the standard run must be kept low. The maximum time allowed will depend on

local conditions and should be kept under review; however, under stable conditions, one

standard run per day should suffice, allowing a single standard run to be applied to several

different samples.

The standards used contain a mixture of oligomers of glucose (derived by partial acid

hydrolysis of dextran) or arabinose with elution times covering the whole range inwhich sample peaks are expected. Standard mixtures of glycans are sold for the purpose

(Prozyme, Ludger). These have been defined above and contain linear oligomers of all

lengths up to and beyond 20, in consistent proportions. Chromatograms produced from

such mixtures take the form of a ladder, as shown in Figure 12.6.18.

Two standard scales are definedthe glucose unit (GU) scale for NP-HPLC and the

arabinos unit (AU) scale for RP-HPLCeach based on the glycan chain length of

standard linear oligosaccharides. These scales both employ a least-squares fifth-order

polynomial curve-fitting algorithm. The GU value of a glycan is directly related to the


30/45


12.6.30


Figure 12.6.18 Standard ladder chromatograms and calibration curves for (A) dextran and (B)arabinose. Abbreviations: AU, arabinose units; GU, glucose units.

number and linkage of its constituent monosaccharides. Thus GU values can be used to

predict structures because each monosaccharide in a given linkage adds a set amount to

the GU value of a glycan. No direct correlation between AU value and structure has yet

been determined.

Calibration of the HPLC System

1. Select a region of the ladder to use, such that each constituent ladder peak is clearlydefined and distinguished from impurity peaks.

The region should be as large as possible; however, peaks at the ends of the ladder should

not be used if there is any difficulty in assigning them. It is very important to include thewhole of the region in which sample peaks are expected. The highest reproducibility will

be obtained if the same region is used on every occasion.

2. Label the ladder peaks within the chosen region with integer values representing the

associated glycan chain length. Check once again that the correct peaks have been

identified, and that the numbers are consecutive and correctly represent the glycan

chain lengths.

3. Create a calibration curve by applying a least-squares fifth-order polynomial curve-

fitting algorithm to the pairs of data, treating the elution time as the independent

variable (xaxis) and the integer values as the dependent variable (yaxis).

This calibration curve is then used to determine the GU/AU values for the sample peaks.

4. If using Waters GPC software, create an apex track GPC processing method with a

relative fit type of fifth order and a calibration order of molecular weight low to high.

This method will then generate the GU (or AU) values and store them with the rest of thepeak data.

Examples of GU and AU calibration curves are shown in Figure 12.6.18.


31/45

Glycosylation

12.6.31


BASIC

PROTOCOL 7

SEQUENCING AN ENTIRE N-GLYCAN POOL SIMULTANEOUSLY USINGEXOGLYCOSIDASE ARRAYS

In this protocol, the N-glycan pool is digested with specific exoglycosidases to establish

an array of digestion patterns. Enzyme specificities and digest conditions are noted in

Tables 12.6.4 and 12.6.5, and a schematic representation is given in Figure 12.6.19.

The glycan pool is profiled by NP-, RP-, and WAX-HPLC (see Basic Protocol 6) and

MALDI-MS (UNIT12.7). If sample is limited, use only NP-HPLC, as this will give the

most information for the minimum amount of material.

Figure 12.6.20 and Table 12.6.6 show data for sequencing of the N-linked glycans of

human neutrophil gelatinase Bassociated lipocalin (Rudd et al., 1999a).

Materials

N-glycan pool (2-AB labeled; see Basic Protocol 5)

Exoglycosidase enzymes and buffers (Table 12.6.4)

200-l microcentrifuge tubes

Speedvac evaporator

37C incubator or water bath.

Micropure-EZ centrifugal filter devices (Millipore)

Additional reagents and equipment for NP-HPLC (see Basic Protocol 6)

Perform enzymatic digests

1. Pipet aliquots of the entire N-glycan pool into 200-l microcentrifuge tubes and

evaporate to dryness using a Speedvac evaporator.

2. Perform enzymatic digest as described in Table 12.6.4, following the arrays set up

in Table 12.6.5, in a volume of 10 to 20 l, including buffer. Digest 16 hr at 37C

in a water bath or incubator. For example, for a triple digest with ABS, AMF, and

BTG, use:

1 l ABS

1 l AMF

2 l BTG2 l 5 incubation buffer (250 mM sodium acetate, pH 5.5)

4 l water.

If using a water bath, gently centrifuge the contents to the bottom of the tube from time to

time, so that water condensing on the lid does not cause the concentrations in the digestto change. In the case of combined digests, use the average optimum pH conditions of theconstituent enzymes.

All enzyme digests should be carried out with reference to the suppliers literature. Theenzymes noted in Table 12.6.4 are supplied by Prozyme (or, alternatively, Seikagaku

Kogyo, Roche, Merck, and Sigma). Full technical information and storage conditions canbe found in the manufacturers information and catalogs.

3. Remove reaction vial from the incubator or water bath and briefly spin down the

contents to the bottom of the tube.


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Oligosaccharides

N-andO-Linked

toGlycoproteins

12.6.32

Supplement43

CurrentProtoco

lsinProteinScience

Table 12.6.4 Conditions for Digestion of Glycan Pools by Exoglycosidase Enzymes

Enzymea Specificityb Make up as % of final

volcFinal enzyme

concentration F

ABS Sialic acid 2-6> 3,8 0.2 U + 20 l water 10% 1 mU/ l 100 mM

NDVS Sialic acid 2-3, 8 20 mU + 10 l water 10% 200 U/l 50 mM

BTG Gal 1-3, 4> 6 As is 20% 1 mU/ l 100 mM

SPG Gal 1-4 40 mU + 100 l water 20% 80 U/l 100 mM

AMF Fuc 1-3, 4 20 U + 2 l water 10% 1 U/l 50 mM

BKFd-i

Fuc 1-6 (>23,4) 0.1 U + 10 l water 10% 1 mU/ l 100 mMJBHd,j,k GlcNAc, GalNAc 1-2, 3, 4, 6 As is 20% 10 mU/ l 100 mM

SPHd,f,m,n GlcNAc1-4, 6 GlcNAc1-3,6

Gal

30 mU + 100 l water 40% 120 U/l 100 mM

JBM (high)d,j,o Man 1-2, 3, 6 2 U in 30 l 1 buffer 100% 67 mU/ l 100 mM

JBM (low)d,j Man 1-2, 6 2 U in 30 l 1 buffer 8% 5.4 mU/ l 100 mM

HPM Man 1-4 GlcNAc 0.2 U in 10 l water 10% 2 mU/ l 100 mM

aThis table shows most commonly used enzymes. For further details refer to manufacturers inserts. Abbreviations: ABS, Arthrobacter ureafacienssialidas

-fucosidase; BTG, bovine testes -galactosidase; HPM,Helix pomatia -mannosidase; JBH, jack bean -N-acetylhexosaminidase; JBM, jack bean -m

SPG,Streptococcus pneumoniae -galactosidase; SPH,Streptococcus pneumoniae-hexosaminidase.bSee manufacturers inserts for further details.c

Also includes 20% 5 incubation buffer.d-nThe following notes refer to inhibitory metal ions: dHg2+.e L-fuctose;fNi2+;gCu2+;h Ag2+;i 2 M 4-chloromercuriphenylsulfonic acid;jAg+;kFe3+;l CoAdd second aliquot at 12 to 18 hr.


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Glycosylation

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Table 12.6.5 Exoglycosidase Arrays Commonly Used in N-Glycan Sequencing

Enzyme 1 2 3 4 5 6 7a

ABS X X X X X

NDVS X

AMF X X X X

BTG X X X

BKF X X

SPH X

JBM (high) X

aJBM is used only when the presence of oligomannose structures is indicated, i.e., structures

resistant to other enzyme arrays.

Figure 12.6.19 Diagram showing specificities of exoglycosidases. For enzyme abbreviations,see Table 12.6.4.

Remove enzymes

4. Prewash a Micropure-EZ centrifugal filter device with 200l of water by microcen-

trifuging 10 min at 7000 g, room temperature. Discard wash.

The sample is filtered to remove the majority of enzymes and the carrier protein, BSA,

in order to prevent contamination of the HPLC column. However, sialidase activity hasbeen noted on the columns following many analyses of enzyme digestions. Filtering the

samples prior to HPLC is good laboratory practice.

5. Add the sample to the filter and microcentrifuge 2 min at maximum speed, roomtemperature.

6. Wash out the sample tube with 20l of water, apply to the filter, and microcentrifuge

2 min at maximum speed, room temperature.

7. Add 100 l of water to the filter and microcentrifuge 5 min at maximum speed, room

temperature.

8. Dry down the sample and dissolve in 20l water. Add 80l acetonitrile and inject

onto an NP-HPLC column (see Basic Protocol 6).


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12.6.34


Figure 12.6.20 Sequencing N-linked glycans from neutrophil gelatinase Bassociated lipocalin.For enzyme abbreviations, see Table 12.6.4.


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Glycosylation

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Table 12.6.6 HPLC Analysis of Exoglycosidase Digestions of the PNG-released, 2-AB-labeled Pool of NGAL N-Glycans

Digests (GU value)

Peak

% Assigned

undigested

peaks Assignment

Undigested

peaks

(GU value) ABS ABS/BTG

ABS/BTG/

BKF

ABS/BTG/

BKF/AMF

1 A2 5.50 5.50 5.53

2 F(6)A2 5.93

3 A2FG1 7.07 7.06

4 A2G2 7.15

5 F(6)A2FG1 7.50

6 F(6)A2G2 7.57

7 14.7 A2FG2 7.96 7.96

8 6.2 A2G2S2 8.28

9 8.9 F(6)A2FG2 8.39 8.38

10 38.2 A2FG2S 8.64

11 32.0 F(6)A2FG2S 9.04

9. Assign GU values to the NP-HPLC profiles of the digests and stack them in order

(1 to 6) so that the changes in the elution position of each peak can be followed from

the initial profile through the different enzyme digests.

Peaks are assigned from a knowledge of the specificity of the enzymes in the arrays(Table 12.6.6) and the incremental values of individual monosaccharide residues (Table

12.6.1 Guile et al., 1996). The final enzyme array for N-glycan pools is designed todigest all the oligosaccharides to their basic cores. This means that sugars containing

less-common oligosaccharides such as xylose, or sugars containing substitutions such assulfate or phosphate, will be only partially digested and can be recovered and examined

independently.

BASIC

PROTOCOL 8

SEQUENCING O-GLYCANS

This protocol represents the authors current progress in developing HPLC-based meth-

ods for analyzing O-glycosylation. O-linked glycans are more complicated to sequence

than N-linked sugars. This is partly because the heterogeneity can be very complex, par-

ticularly in glycan pools released from mucins (Fig. 12.6.21). In addition, O-linked sugars

are constructed from eight different core structures and have more complicated branching

patterns than N-glycans. Of the O-linked core structures listed in Figure 12.6.7, four are

diHexNAc and two are HexHexNAc, so mass alone cannot be used to identify them.

The elution positions of these disaccharides on NP-HPLC are very similar, so RP-HPLC

is required as a complementary separation method. A greater range of exoglycosidases

than is currently available is needed to determine the branching of the O-glycans. Theenzymes routinely used for N-link sequencing can all be used, but it is important to

test out their specificity on standard O-linked glycans first. Mass data from MALDI or

quadrupole time-of-flight (Q-TOF) analysis and fragmentation data from Q-TOFby

on-line liquid chromatography MS (LC-MS) or single injectionsmay also be needed

to complement HPLC data to enable structures to be assigned (Royle et al., 2002).

Figure 12.6.16 shows the GU and AU values for a range of O-glycans. The incremental

values for individual monosaccharide linkages are similar to those for N-glycans, and

can be used to aid in the assignment of new O-glycans (Royle et al., 2002).


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12.6.36


Figure 12.6.21 Profile of 2-AB-labeled glycan pool released from porcine stomach mucin.

The strategy for O-glycan analysis is shown in Figure 12.6.22. Sequencing the released

glycans involves essentially the same enzymes as those used for N-linked glycans, but

the arrays are different.

Materials

Exoglycosidase enzymes (Table 12.6.4)

Chicken liver-N-acetylhexosaminidase (CLH)

Additional reagents and equipment for HPLC (see Basic Protocol 6) and arrays ofenzymatic digests (see Basic Protocol 7)

1. Resolve samples of the glycan pool by NP-, RP-, and WAX-HPLC (see Basic

Protocol 6, Fig. 12.6.23). Assign preliminary structures to peaks on the basis of GU

and AU values.

2. Take aliquots of the glycan pool and digest with the appropriate enzymes (see Basic

Protocol 7 for procedure; see Table 12.6.7 for enzyme array). Analyze the digestion

products by NP- and RP-HPLC.

3. If ABS digests, digest with NDVS.

If ABS digests but NDVS does not, sialic acid is linked2,6.

4. If BTG digests, digest with SPG.

If this also digests, galactose is linked1,4.

5. If BKF digests, digest with AMF.

If BKF digests but AMF does not, fucose is 1,2 linked.

6. If JBH digests, digest with SPH.

SPH digests only GlcNAc, whereas JBH digests both GalNAc and GlcNAc.

Figures 12.6.24 and 12.6.25 show a selection of these digests, which enable the O-glycansattached to fetuin to be analyzed.


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Glycosylation

12.6.37


Figure 12.6.22 A strategy for the analysis of O-glycans.

Table 12.6.7 Exoglycosidase Arrays Commonly Used in O-Glycan Sequencing

Enzyme 1 2 3 4 5 6 7

ABS X X X X

BTG X X

BKF X X

JBH X X

REAGENTS AND SOLUTION

Use Milli-Q-purified water or equivalent for the preparation of all buffers. For common stocksolutions, see APPENDIX2E; for suppliers, see SUPPLIERS APPENDIX.

Buffer A

5 ml 2 M ammonium formate, pH 4.4

50 ml Milli-Q-purified water

Adjust pH to 8.6

Add 0.4 g SDS

Bring to 100 ml

Store up to 6 months at 4C


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12.6.38


Figure 12.6.23 Resolution of the O-glycans attached to fetuin using NP-, RP-, and WAX-HPLC.


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Glycosylation

12.6.39


Figure 12.6.24 Sequencing fetuin O-linked glycans using NP- and RP-HPLC. For enzyme abbreviations, see Table12.6.4.

Buffer B

5 ml 2 M ammonium formate, pH 4.4

50 ml Milli-Q-purified water

Adjust pH to 8.6

Add 1.2 g CHAPS (1.2% w/v)

Add 3.72 g EDTA (0.1 M)

Bring to 100 ml

Store up to 6 months at 4C

HPLC standards

Dextran ladder (NP-HPLC): 2-AB labeled homopolymer stranded (Prozyme,

Ludger)

Arabinose ladder (RP-HPLC):Label individual arabinose homopolymers with 2-

AB and co-inject onto the RP column.

Arabinose is available from Fluka, and arabinobiose to arabinooctaose forms are availablefrom Dextra Laboratories.

Fetuin N-linked glycans (WAX-HPLC):Label fetuin N-linked glycans (Prozyme,

Ludger) with 2-aminobenzamide.

The mono-, di-, tri-, and tetrasialylated glycans are separated by WAX-HPLC.


40/45


12.6.40


Figure 12.6.25 Structures, GU values, and AU values corresponding to peaks in Figure 12.6.24. For enzymeabbreviations, see Table 12.6.4.

SDS sample buffer, 5

625 l 0.5 M TrisCl, pH 6.6 (APPENDIX 2E)

1.0 ml 10% (w/v) SDS (APPENDIX 2E)2.875 ml 10% glycerol

0.4 g bromphenol blue

Store up to 1 year at 20C

Solvent A ( for NP-HPLC)

Stock solution (2 M formate): Prepare a 2-liter 2 M formate stock solution as for

solvent E (see recipe), but adjust pH to 4.0 with ammonia (260 ml), allow to

cool and settle outside the ice bath, and then adjust to pH 4.4.

Working solution (50 mM): Dilute 50 ml stock to 2 liters using purified water.

Prepare fresh.

Solvent C (for RP-HPLC)

Stock solution (1 M formate): Prepare a 1 M formate stock solution as described

for solvent E (see recipe), but start with 92.06 g formic acid and adjust pH to 5 with

triethylamine (BDH HiPerSolv for HPLC), which acts as an ion-pairing agent.


Prepare fresh.


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Glycosylation

12.6.41


Solvent E (for WAX-HPLC)

Stock solution (2 M formate): Place a 2-liter beaker in an ice bath containing salt

to lower the temperature of the ice to 10C. Weigh 184.12 g formic acid (BDH

Aristar grade) into the beaker using a glass acid-washed pipet. Turn off the fume

hood while weighing, since air flow will affect the balance. Add 1 liter water

while stirring. Adjust pH to 8.5 bydropwiseaddition of 35% ammonia solution

(BDH Aristar grade). Remove beaker from ice and allow the warm solution to

cool to room temperature (1 hr). Adjust pH to 9.0 with ammonia solution, added

dropwise. Transfer solution to a 2-liter volumetric flask, bring to volume using

purified water, and note the final pH. Transfer to a brown bottle and store up to

several months at room temperature.


Check pH before using. Prepare fresh.

CAUTION:DO NOT add ammonia solution in large quantities as this will produce a rapid

and potentially dangerous increase in temperature. When using formic acid or ammonia,place the pH meter and balance in an empty fume hood. Wear a laboratory coat, gloves,

and goggles.

COMMENTARY

Background InformationIn recent years, major advances in thetechnology available for separating and se-

quencing oligosaccharides from glycoproteins

have brought glycan analysis of micromolar

amounts of biological samples within thereach

of any well-founded laboratory. It is now pos-

sible to release N-linked oligosaccharides di-

rectly from 5 to 30 g of protein in an SDS-

PAGE gel band and analyze them by mass

spectrometry (UNIT 12.7) or HPLC (Basic Pro-

tocol 6). HPLC strategies allow structures of

subpicomolar levels of neutral and sialylated

oligosaccharides to be predicted from a singlerun, while further information can be obtained

rapidly using multiple exoglycosidase arrays

to analyze all of the components of a glycan

pool simultaneously.

When the three-dimensional structure of

the protein and oligosaccharide analysis are

both available, the new database of premini-

mized oligosaccharide structural units can be

assembled appropriately and modeled onto the

protein. The justification for this stems from

the fact that, in contrast to proteins, where ter-

tiary and quaternary interactions play a sig-

nificant role, the main structural features ofoligosaccharides are determined by their sec-

ondary structure, which involves interactions

across individual glycosidic linkages.

Molecular modeling of glycoproteins re-

quires an oligosaccharide analysis of the sug-

ars associated with the protein. However, the

classical strategies and techniques used to lo-

cate glycans and separate and analyze the

oligosaccharides released from the heteroge-neous mixture of glycoforms (for review, see

Dwek et al., 1993; Rudd et al., 2001), although

effective, were time consuming and difficult.

Moreover, the poor resolution achieved by the

separation technologies and the insensitivity

of the detection systems precluded the analy-

sis of the glycosylation of many biologically

important glycoproteins that are only available

in microgram quantities or less.

It is important to retain a flexible and in-

teractive approach to sequencing strategies to

obtain specific information required to answer

particular biological questions.However, thereis also a need for a robust technology that is

generally applicable to all glycoproteins, so

that oligosaccharide sequencing at the subpi-

comolar level can become as rapid and au-

tomated as peptide sequencing, and routinely

available to protein chemists as well as gly-

cobiologists. It is such a technology that is

described in this unit and in UNIT 12.7, which

is based solely on methodology used at the

Glycobiology Institute.

Critical Parameters and

TroubleshootingWhen carrying out procedures on sam-

ples that might be analyzed by mass spec-

trometry (UNIT 12.7), it is essential to use the

highest-quality solvents for every purpose. In

particular, purified water sometimes contains

polymeric material (principally polyethylene

glycol) that will co-concentrate with the sugars

and impair MALDI-MS detection. To avoid


42/45


12.6.42


this problem, purified water should be further

treated, e.g., by distilling at sub-boiling-point

temperatures (SBPD) to prevent organic con-

taminants from being carried across with the

steam. For purposes where purified water is

adequate the quality should be total organic

carbon (TOC)


43/45

Glycosylation

12.6.43


Glycans should not be left dried in tubes

for long periods, as they may subsequently be

hard to remove from vial/tube walls.

Anticipated ResultsRepresentative chromatograms and profiles

are presented in figures within each of the spe-

cific protocols. The structures should be con-

sistent with known biosynthetic pathways. If

not, care must be taken to confirm the assign-

ments, as novel structures may imply novel

pathways.

Time ConsiderationsThe time required for each protocol is be-

low. Figure 12.6.10 offers additional informa-

tion on interrelation of the protocols. PNGase

F release of N-linked glycans (Basic Proto-

col 1) requires 2 days. In-gelenzymaticrelease

(Basic Protocol 2) will also take 2 days, as will

the identification of glycoproteins through in-

gel trypsin digestion (Basic Protocol 3). Man-

ual hydrazinolysis (Basic Protocol 4) can takeanywhere from 5 to 8 days to complete.

Fluorescent labeling of theglycan pool with

2-AB (Basic Protocol 5) takes 3 to 5 hr. HPLC

separation (Basic

determining the structure of unit 12.6 oligosaccharides n- and o-linked to glycoproteins

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