Proc. Natl. Acad. Sci. USAVol. 85, pp. 3289-3293, May 1988Biochemistry

Separation of positional isomers of oligosaccharides andglycopeptides by high-performance anion-exchange chromatographywith pulsed amperometric detection

(HPLC/electrochemical methods/complex carbohydrates/bovine fetuin)

MARK R. HARDY AND R. REID TOWNSEND*Department of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, MD 21218

Communicated by Christian B. Anfinsen, January 6, 1988

ABSTRACT High-performance anion-exchange (HPAE)chromatography under alkaline conditions (pH ~=13) has beenfound to efficiently separate neutral oligosaccharides (triose toundecaose) according to molecular size, sugar composition,and linkage of monosaccharide units. The method was able toresolve 1 -* 3, 1 -> 4, and 1 -* 6 positional isomers of neutraloligosaccharides, which are dermed as having the same num-ber, type, sequence, and anomeric configurations of mono-saccharides but differing in the linkage position of a singlesugar. From correlating structural features of different oligo-saccharides and retention times, we deduced that at least twofactors are operative to determine the superior resolution ofoligosaccharides by this type of chromatography: (i) therelative acidities ofthe hydroxyl groups and (ii) the accessibilityof oxyanions of the oligosaccharides to the functional groups ofthe stationary phase. Splitting of peaks attributable to muta-rotation was not observed. Reducing oligosaccharides weremuch more retained than their reduced counterparts. Linkageof Fuc(al-3) to GlcNAc of oligosaccharides markedly de-creased retention times. Positional isomers of two branchednonosaccharides, which differed by 1 -*6 and 1 -*4 linkages,were widely separated. The separation of 1 -> 3 and 1 -* 4positional isomers of both tetrasaccharides and glycopeptidescontaining undecasaccharides demonstrated the significantimprovement in resolution of HPAE compared to previouschromatographic methods by either reverse-phase or amine-bonded stationary phases. Picomole quantities of underivatizedoligosaccharides have been detected by triple-pulse amper-ometric detection, which produced similar responses for a widerange of structures. Quantification of two triantennary glyco-peptides from bovine fetuin by using either detector responseor 'H NMR was comparable. The N-glycanase-catalyzed re-lease of two 1 -* 4 and 1 -* 3 positional isomers of anundecasaccharide from a tryptic glycopeptide of bovine fetuincould be observed and quantified by direct injection of theenzyme mixture into the chromatograph.

Complex carbohydrates have been implicated in a variety ofbiological reactions. Cell-cell recognition in development (1)and cancer metastasis (2), intracellular transport oflysosomalenzymes (3), and antibody reactivity to soluble and cell-bound carbohydrate determinants (4) are some well-studiedexamples. Studies designed to elucidate the structural basisof the biological reactivity of naturally derived carbohydratesfrequently require the resolution of complex mixtures ofoligosaccharides (usually from glycoproteins) or glycolipids.Separation of oligosaccharides that vary only by a singlelinkage position is often required to define specificities ofantibodies (5, 6), lectins (7, 8), and glycosyltransferases (9,10). We report that high-performance anion-exchange chro-

matography efficiently resolved positional isomers of oligo-saccharides and glycopeptides and coupled to electrochem-ical detection by pulsed amperometry (HPAE-PAD) (11)allowed detection of both reducing and nonreducing underi-vatized carbohydrates in the pmol range.HPLC methods have been developed to separate oligosac-

charides (triose to undecaose) that differ by small structuralfeatures (for review, see ref. 12). Neutral oligosaccharidesdiffering in content by one sugar residue can be resolved byusing either alkyl- (13, 14) or amine-bonded (15, 16) station-ary phases. Positional isomers of neutral oligosaccharides,which are defined as having the same number, type, se-quence, and anomeric configurations ofmonosaccharides butdiffering in the linkage position of a single sugar, are moredifficult to resolve (13, 16). Separations of 1 -* 6 positionalisomers from their 1 -*2, 1 -*3, and 1 -*4 counterparts havebeen achieved for a number of oligosaccharides with - 13monomers (14, 16). However, resolution among 1 -* 2, 1 -*3, and 1 -* 4 isomers has been routinely successful for onlydi-, tri-, and tetrasaccharides (13, 17). We report that HPAE-PAD chromatography at alkaline pH separated not only 16 positional isomers but also resolved isomers (4-11 mono-saccharide units) and glycopeptides differing only in a singleGal,3(1,3) versus a Gal,8(1,4) linkage.With the exception of relatively insensitive detection

methods using refractive index and intrinsic UV absorbance(reviewed in ref. 12), derivatization of oligosaccharides,usually at the reducing end, with chromophores (18, 19),fluorophores (14), or radioactive isotopes (20), is required toattain practical sensitivity. We show that pulsed amperomet-ric detection has a sensitivity range of 10-100 pmol for botholigosaccharides and glycopeptides.The analysis of oligosaccharides linked to glycoproteins

has been enhanced recently with the introduction ofenzymessuch as N-glycanase [peptide-N4-(N-acetyl-,B-glucosami-nyl)asparagine amidase] (14, 21), which can cleave a widerange of oligosaccharide structures from protein (14, 22-24).However, a single-step quantitative assay to monitor thedisappearance of substrate (e.g., glycopeptide or glycopro-tein) and the appearance of resolved oligosaccharides has notbeen developed. The coupling of pulsed amperometry, whichcan detect both reducing and nonreducing carbohydrate, to achromatographic method that can potentially resolve alloligosaccharide isomers would permit sensitive, rapid "oh-gosaccharide mapping" of either glycoproteins or individualglycosylation sites. We show that the N-glycanase-catalyzedrelease of two 1 -* 4 and 1 -* 3 positional isomers of anundecasaccharide from a tryptic glycopeptide of bovine

Abbreviations: HPAE-PAD, high-performance anion-exchangechromatography with pulsed amperometric detection; RP, reversephase; AB, amine-bonded; TRI, triantennary glycopeptide.*To whom reprint requests should be addressed.

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fetuin was observed and quantified by direct injection of theenzyme mixture into the chromatograph.

MATERIALS AND METHODSMaterials. Many of the compounds used in this study were

prepared by the following individuals (the number corre-sponds to the designation of oligosaccharides in Table 1): 1,3, 10, 12, and 13, Hans Lonn (Department of OrganicChemistry, University of Stockholm); 2, 6-8, 9, 11, 14-16, J.Lonngren (Department of Organic Chemistry, University ofStockholm); 5, V. Ginsburg (National Institutes of Health).Compound 4 was purchased from BioCarb (Lund, Sweden).Neuraminidase from Arthrobacter ureafaciens was kindlyprovided by Y. Uchida (Uji, Japan). N-Glycanase [peptide-N4-(N-acetyl-,B-glucosaminyl)asparagine amidase] was fromGenzyme (Boston). Asialotriantennary glycopeptides from aPronase digest of bovine fetuin were prepared and charac-terized as described (8). Trypsin was from WorthingtonBiochemicals (Malvern, PA) and Pronase was from Calbio-chem. NaOH solution (50%) (wt/wt) was purchased fromFisher. Sodium acetate was from J. T. Baker (Philipsburg,N.J.). Nylon membranes were from Schleicher & Schuell.

Preparation Qf Tryptic Glycopeptides. Asialoglycopeptidesfrom reduced and alkylated bovine fetuin were prepared bydigestion with L-1-tosylamido-2-phenylethyl chloromethylketone-treated trypsin, digestion with neuraminidase from A.ureafaciens, and chromatography on DEAE-Sephacel essen-tially as described (36). A glycQpeptide from the Asn-Gly sitewith an amino acid sequence of Ser-Asn-Gly-Ser-Tyr-Leuwas purified by reverse-phase (RP)-HPLC as described (36).Chromatographic Apparatus. The system used for HPAE-

PAD consisted of a Dionex (Sunnyvale, CA) Bio LC gradientpump and model PAD 2 detector. The Dionex Eluant DegasModule was used to sparge and pressurize the eluants withhelium. For gradients 1 and 2, eluant A was 100 mM NaOHand eluant B contained 100 mM NaOH containing 0.15 Msodium acetate. Eluant B contained 0.5 M sodium acetate forgradient 3. These solutions were prepared by suitable dilutionof a 50% NaOH solution with glass-distilled water. Sodiumacetate-containing eluants were filtered through 0.2-1Lm ny-lon membranes before use. Gradient 1, used for the separa-tion of neutral oligosaccharides, was 10 min of isocraticelution with eluant A followed by a linear increase in eluantB (up to 80o at 60 min). Gradient 2, used for the separationof less acidic asialoglycopeptides, was linear to 150 mMsodium acetate over 65 min after a 2-min elution with eluantA. Equilibration was accomplished by a 20-min elution with0.1 M NaOH. Gradient 3 was produced by the followingchanges in concentration of eluant B: 0 min, 0%; 2 min, 0%;60 min, 30%; 118 min, 60%; 120 min, 60%; 125 min, 0%; 130min 0%.Sample injection wastVia a Spectra Physics (Santa Clara,

CA) SP8780 autosampler equipped with a 200-,u1 sample loop.The Rheodyne injection valve on the autosampler wasequipped with a Tefzel rotor seal to withstand the alkalinityof the eluants. Oligosaccharides and glycopeptides wereseparated on a column (4.6 x 250 mm) of Dionex CarboPacPA-1 pellicular anion-exchange resin with a flow rate of 1ml/min at ambient temperature. For most experiments, aCarboPac PA guard column (3 x 25 mm) was also used.NaOH (300 mM) was added to the postcolumn effluent via amixing tee at a flow rate of 1 ml/min with the Dionex Auto-ionReagent Pump. Detection was by PAD with a gold workingelectrode and triple-pulse amperometry (11, 25). The follow-ing pulse potentials and durations were used for detection ofoligosaccharides: E1 = 0.05 V (t1 = 360 ms); E2 = 0.80 V (t2= 120 Ms); E3 = -0.60 V (t3 = 420 ms). The response timeof the PAD 2 was set to 3 s. Chromatographic data were

collected and plotted by using either Spectra Physics modelSP4270 integrator or a Waters model 840 software.N-Glycanase Digestion. Tryptic glycopeptide (50 nmol) was

dissolved in 740 Al of 0.25 M sodium phosphate (pH 8.4).N-Glycanase (1.5 mu, 1.5 Genzyme units) was added and themixture was incubated at 370C. At various times, aliquots (=1nmol) of the digest were chromatographed as describedabove on the AS-6 column with no guard column.

RESULTS AND DISCUSSIONThe hydroxyl groups ofcarbohydrates have pKa values in therange of 12 to 14 (26) enabling ionization at alkaline pH andpotential separation by anion-exchange chromatography.The anomeric hydroxyl group of reducing sugars is muchmore acidic than the other ring hydroxyl groups, which havea hierarchy of acidity of 2-OH >> 6-OH > 3-OH > 4-OH(26). This difference in acidity of hydroxyl groups for mono-saccharides and the consequent expected changes in acidityof oligosaccharides containing different linkages suggests ahigh potential for chromatographic selectivity. Indeed, ion-exchange resins in the hydroxide form have been used forseparation of anomers of glycosides (27-29).

Fig. 1 shows the separation of 13 oligosaccharides, rangingfrom tri- to undecaose, by using HPAFi-PAD. Table 1 givesthe structures and retention times of these and 3 additionaloligosaccharides, all of which are related to structures com-monly found N-linked to glycoproteins. Larger oligosaccha-rides, in general, showed greater retention times, which isexplainable on the basis of an increasing number of negativecharges on the oligosaccharides due to increasing numbers ofhydroxyl groups. However, we also found that this type ofchromatography was highly selective for linkage positionsand component monosaccharides. From correlating struc-tural features and retention times, we deduced that at leasttwo factors are operative in determining the superior reso-lution of oligosaccharides by this type of chromatography: (i)the relative acidities of the hydroxyl groups and (ii) theaccessibility of oxyanions of the oligosaccharides to thefunctional groups of the stationary phase.

All oligosaccharides except compound 6 possessed areducing terminal, yet the complication of peak doubling dueto a and (3 anomeric pairs was not observed. Mutarotation,often observed in RP-HPLC (30) and not in amine-bonded(AB)-HPLC (31), is apparently too rapid under these basicconditions. The contribution of the high acidity of theanomeric hydroxyl to retention time is suggested by com-parison of compounds 6 and 7. The conversion of compound7 to the sugar alcohol (compound 6) resulted in a 15.4-min

(Ar_0C.

E

P-0)

IQ'e

.E

Time, min

FIG. 1. Separation of neutral oligosaccharides using HPAE-PAD. A mixture of oligosaccharides (=1 nmol each) were chroma-tographed on a Dionex CarboPac PA-1 column (4.6 x 250 mm) anddetected by PAD. Gradient 1 was used to elute the larger oligosac-charides and is shown by the dashed line.

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Structures and retention times of neutral oligosaccharides by HPAE-PADRetention Com-

Oligosaccharide structure time, min poundFuc(al-3)GlcNAc(J31-2)Man

Gal(31-4)GlcNAc(,81-6)Man

Gal(,31-4)GlcNAc(131-2)Man/

Fuc(al-3)

3.0

10.0

Oligosaccharide structure

Fuc(al-3)GlcNAc(J31-2)Man(al-6)

12

Fuc(al-3)GlcNAc(J31-2)Man(al-3)4.7

Fuc(al-3)

4 Gal(J31-4)GlcNAc(q31-3)Gal(ql-4)Glc

5 Gal(,81-3)GlcNAc(,81-3)Gal(131-4)Glc

Gal(131-4)GIcNAc(131-2)

Man-OH

Gal(,61-4)GlcNAc(q1-2)Man(al-6)18.1

1326.5

Man 9.2

Gal(q3l-4)GIcNAc(j31-2)Man(al-3)

Fuc(al-3)7.8

/al~a1-4)GlcN~c(,B1-4) Gal(131-4)GlcNAc(f,1-2)Man(al-6)Galq,81-4)GlcNAcq,81-4)14

Gal(J31-4)GlcNAc(q1-2)

Man

Man 35.4

Gal(J31-4)GlcNAc(Bl-2)Man(al-3)23.2

Gal(81-4)GlcNAc(f31-4)Gal(,8l-4)GlcNAc(ql8-4)

Gal(,81-4)GlcNAc(p1-2) Gal(f3-4)GlcNAcq8l-6)Man/

Galqfil-4)GlcNAc(q3l-6)

Gal(f31-4)GlcNAc(,31-2)Man(al-6)24.2

15 Man 37.3

Gal(31-4)GlcNAc(J81-2)Man(al-3)Gal(31-4)Glc(,1-2)

Man/

Gal(f31-4)Glc(J31-6)

32.7 Gal(,81-4)GlcNAc(q1-6)

Gal(q1-4)GlcNAc(f31-2)Man(al-6)

16Gal(p1-4)GlcNAc(J31-6)

Man(al-2)Man/

Gal(131-4)GlcNAcqpl-3)

Gal(31-4)GlcNAc(q1-2)Man(al-6)

Man

28.7

Man 39.4

Gal(/31-4)GlcNAc(,8l-2)Man(al-3)/

Gal(f31-4)GlcNAc(,Bl-4)

33.0

Gal(31-4)GlcNAc(q1-2)Man(al-3)

decrease in retention time (Table 1). It is also possible that achange in conformation by conversion of the branchingmannose from a pyranose chair form to a straight chaincaused the decrease in retention time. Although it is prema-ture to generalize to all oligosaccharide structures, we havefound that the alditols of a series of disaccharides also hadmarkedly shortened retention times compared to their reduc-ing counterparts (unpublished data).The most acidic proton in unsubstituted glycosides is the

2-OH due to its proximity to the electron-deficient anomericcarbon (27). In 2-acetamido-2-deoxypyranosides, the 3-OH isrelatively acidic as a result of the acetamido function (27).

Pentasaccharides 8 and 9 differ only in that glucose issubstituted for GlcNAc in both branches of compound 8. Theglucose-containing pentasaccaride had a significantly greaterretention time (8.5 min), suggesting that either the 2-OH ofglucose is more acidic than the 3-OH of GlcNAc or that the2-OH of glucose is more accessible to the surface of thepellicular resin.The effect of substitution of the 3-OH of GlcNAc with

fucose on retention time can be seen with compounds 1, 2,and 3 and compounds 11, 12, and 13. Compound 1, which hasFuc(al-3) substituted for Gal(f31-4) in compound 2, had a7-min shorter retention time. Addition of Fuc(al-3) to com-

Table 1.

Com-pound

1

2

3

Retentiontime, min

Man 5.6

6

7

8

9

10

11

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pound 2, to form a tetrasaccharide, only increased theretention time by 1.7 min. Even though fucose is a 6-deoxysugar, its 2-OH, 3-OH, and 4-OH should compensate, interms of net charge, for the inability to form the 3-OHoxyanion. Marked shortening of retention time by the linkingof Fuc(al-3) to GlcNAc was more impressive for thebranched compounds 11, 12, and 13. Compound 11 is abiantennary structure terminating in Gal(l31-4) linked toGlcNAc. Replacement ofthe termini with Fuc(al-3) removesthe effect of the 3-OH proton of the GlcNAc and the 6-OH ofthe galactose and results in a much shorter retention time(27.4 min). Linkage of two Fuc(al-3) to the galactosyltedcompound (no. 11) produced a 23.8-min decrease in retentiontime. Although this substitution would prevent formation ofa 3-OH oxyanion at both branch GlcNAcs, compound 13 nowhas six additional hydroxyl groups, including the two 2-OHsfrom the added fucoses. Molecular modeling using hardsphere exo-anomeric calculations (32) showed that the 2-OHsof both fucoses of compound 13 are =-4 A from the carbonylcarbon of the acetamido group of GlcNAc (B. N. N. Rao andR.R.T., unpublished results). The acetamido groups of theGlcNAcs apparently prevent interaction of the fucose 2-carbon oxyanions with the stationary phase with a resulting23.8-mim decrease in retention time.The greater retention time of compound 15 compared to 14

is an example of chromatographic selectivity for isomericforms. Since 6-OHs are more acidic than 4-OHs, compound14 was expected to be more retained than compound 15. Thegreater rotational freedom imparted by the 1,6 linkage ap-parently produces a stronger interaction with the stationaryphase. Similarly, in AB-HPLC compound 15 was also re-tained more tightly (16). The greater retention times of 1 -*

6 isomers compared to 1 -> 2, 1 -* 3, and 1 -* 4 counterpartshas been attributed to the enhanced ability of 1 -*6 isomersto form hydrogen bonds with AB columns (16). Alternatively,substitution of the 6 position allows the other OHs of the ringto be more accessible.

Neither AB-HPLC nor RP-HPLC have been shown toefficiently separate underivatized oligosaccharide isomersdiffering only in 1 -* 2, 1 -* 3, and 1 -*4 linkages (13, 16, 17).For example, lactose N-tetraose (compound 5) and lactose-N-neotetraose (compound 4) are extremely difficult to purifyand usually require derivatization and/or additional chroma-tography (13, 17). Pentasaccharides differing only in a

Gal(,81-3) versus a Gal(,81-4) linkage gave identical retentiontimes with AB-HPLC (16).

Fig. 1 and Table 1 show that lactose-N-tetraose andlactose-N-neotetraose are separated by 8.4 min. Assuming agreater acidity for the 3-OH of GlcNAc relative to the 4-OH,we expected that compound 4 would be a more acidicoligosaccharide and, therefore, elute after compound 5.Conformational analysis of these two types of linkagesshowed that the disaccharide unit, Gal(,81-3)GlcNAc, has adistinct hydrophilic face produced by both monosaccharides(33, 34). In contrast for Gal(1-4)GlcNAc, the hydroxylgroups of galactose and GlcNAc are in distinct planes and donot form a "sheet" of hydrophilic residues. We thereforespeculate that these distinct conformational differences al-lowed the Gal(,B1-3)GIcNAc-containing structure to interactmore strongly with the positively charged resin.The separation of these 1 -*3 and 1 -*4 isomers by HPAE

was possible even with glycopeptides containing an undeca-saccharide linked to peptides containing two or three aminoacids. These structures and their retention times are given inTable 2. Compounds 17 and 18, 19 and 20, and 21 and 22correspond to peaks 3, 4, and 8, respectively, in ref. 8. Theseglycopeptides were found to have the same peptide but byNMR analysis were mixtures of the two types of triantennarystructures shown in Table 2 (8). Compounds 17-20 were

separated under the same gradient conditions. It can be seen

Table 2. Separation and quantification of Gal(j31-3)-triantennaryglycopeptide (TRI) and Gal(f31-4)-TRI by HPAE-PAD

Retention time,Compound Glycopeptide min17 R1 45.018 R2 51.019 Rl-Gly 44.820 R2-Gly 50.521 Rl-Cys 37.322 R2-Cys 44.0Ga1(814)GlcNAc(81-2)Man(a1-6) Tyr

Man(814)G1cNAc(pl14)GlcNAc Asn = R1/

Ga(P14)G1cNAc(81-2)Man(a1-3)/

Ga1(J14)G1cNAc(,814)

Ga1(,814)G1cNAc(q1-2)Man(a1-6) Tyr

Man(814)G1cNAc(814)G1cNAc Asn = R2/

Ga1(,814)G1cNAc(81-2)Man(a1-3)/

Compounds 17-20 were separated by using gradient 2 and com-pounds 21 and 22 were separated by using gradient 3.

that the addition of the uncharged amino acid, glycine, doesnot change the retention times significantly. The difference inretention times of Gal(,31-3)-TRI and Gal(,81-4)-TRI is -6 minwith and without glycine. The cysteine-containing glycopep-tide is much more retained and requires a higher concentra-tion of sodium acetate for elution (gradient 3). Despite thegreater affinity of compounds 21 and 22 for the stationaryphase, separation of the two oligosaccharide isomers by =6min still occurred.

Derivatization of oligosaccharides and glycopeptides witheither chromophores (18, 19), a fluorophore (14), or aradioactive isotope (20) has been required for detection in thepmol range. Previous studies have shown that triple-pulseamperometric detection allows detection of mono- and di-saccharides in the pmol range without derivatization (11, 25).Peak currents, varying by -2-fold, were observed for bothreducing monosaccharides as well as for sugar alcohols (11).Similarly, we found that the detector response was the samefor neutral hexoses and fucose, a 6-deoxyhexose. However,amino sugars gave approximately a 20% greater response(35). Interestingly, the detector response for the oligosac-charides shown in Fig. 1 also varied by =20%. Closely relatedcompounds-for example, compounds 14, 15, and 16-gavethe same detector response (within 2-5%).These results prompted us to compare the quantification of

Gal(J1-3)-TRI and Gal(Q31-4)-TRI using either integration ofunique NMR signals or PAD response. Table 3 shows that

Table 3. Quantification of Gal(f31-3)-TRI and Gal(Q1-4)-TRI byusing either NMR or HPAE-PAD

Compound HPAE-PAD NMRt17 64 6418 36 3619 36 3020 64 7021 80 8022 20 20

Structures of the glycopeptides are given in Table 2.tThe proportions are calculated from integration of NMR signalsassigned to the NAc methyl protons of the GlcNAc residues 7 and7* in ref. 8, to which is linked galactose either 81-4 or ,81-3.

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Proc. Natl. Acad. Sci. USA 85 (1988) 3293

~~~~~~~~~~4 h0.~~ ~ ~ ~ ~ ~~~~2

0 20 40 60

Time, minFIG. 2. HPAE-PAD analysis of the time course of deglycosyla-

tion of a fetuin tryptic glycopeptide using N-glycanase. Asialogly-copeptide (50 nmol) was treated with 1.5 units of N-glycanase in 0.25M sodium phosphate. At the indicated times, aliquots containing -1nmol of glycopeptide were removed and chromatographed. Thedashed line shows gradient 2.

both methods gave the same proportions of Gal(q1-3)-TRIand Gal(/314)-TRI for three different glycopeptides. It ispremature to conclude that all neutral and charged carbohy-drates will produce the same detector response, but within agroup of related compounds PAD may enable easy, sensitivequantification of oligosaccharide mixtures.

Structural analysis of the carbohydrates ofglycoproteins isroutinely performed by removal of protein by either enzy-matic (14, 21) or chemical (20) methods. The use of N-glycanase, which releases a wide spectrum ofoligosaccharidestructures, has been particularly useful (14, 22-24). Sinceboth oligosaccharides and glycopeptides can be detected andquantified with PAD, the time course of release of differenttypes of oligosaccharides and the disappearance of glyco-peptide can be directly measured. Fig. 2 shows the timecourse of incubation of a glycopeptide from the Asn-Glyglycosylation site of bovine fetuin. With these amino acids(Ser-Asn-Gly-Ser-Tyr-Leu), the two TRI isomers were notseparated. However, as the oligosaccharides were releasedthe two isomers become apparent after 48 hr and completerelease occurred after 120 hr of incubation. The proportion ofGal(l31-3)-TRI to Gal(pl-4)-TRI in the enzyme mixture wasdetermined to be 6:4, which is in good agreement withprevious NMR data and PAD quantification of glycopeptideisolated from this site by Pronase digestion (Table 3, com-pounds 19 and 20).

In summary, HPAE chromatography at alkaline pH sep-arated a wide spectrum of oligosaccharide structures. Allpositional isomers examined were resolved with a linearsodium acetate gradient. Resolution of different carbohy-drate structures was accomplished in glycopeptides thatcontained undecasaccharides and two or three amino acids.Electrochemical detection enabled pmol level detection ofoligosaccharides and glycopeptides. Direct assay by HPAE-PAD of underivatized oligosaccharides released by N-glycanase should now permit sensitive, efficient analysis ofoligosaccharides on glycoproteins.The insightful discussions with Prof. Y. C. Lee were crucial to the

success of this work. Samples donated by Drs. H. LUnn, J. Lonngren,and V. Ginsburg were kindly provided by Prof. Y. C. Lee. We wish tothank Dr. Joe Olechno, Dr. Steven Carter, and others of the DionexCorporation for their suggestions during the course of this work. Thefruitful discussions ofoligosaccharide conformation with Dr. NarasingaRao is gratefully acknowledged. R.R.T. and M.R.H. were supported byNational Institutes of Health Grant DK31376 and National Science

Foundation Grant DCB8509638. The Dionex Carbohydrate Analyzerwas purchased with National Institutes of Health Research GrantDK09970 to Dr. Y. C. Lee and analyses were performed in hislaboratory. This is contribution no. 1403 from the McCollum-PrattInstitute.

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