Characterization of N-linked oligosaccharides by electrospray and

Anal. Chem. 1992, 64, 1440-1448

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Characterization of N-Linked Oligosaccharides by Electrospray and Tandem Mass Spectrometry Kevin L. Duffin and Joseph K. Welply Momanto Corporate Research, Momanto Company, 700 Chesterfield Village Parkway, St. Louis, Missouri 63198

Eric Huangt and Jack D. Henion' Drug Testing and Toxicology, Cornell University, 925 Warren Drive, Ithaca, New York 14850

Eiectrospray and tandem mass spectrometry are used to characterize underlvatlzed oligosaccharides that have been dlgested from asparagine side chrlns of glycoprotelns. 011gosaccharldes that contain sialic aclds were detected with the best sendtlvttyInthe nqatlve-ion detection mode whereas those that do not contain siailc acld were detected with the best sensltlvltyinthe porltlve-iondetection mode. The poOnhre ion abundance8 of oligosaccharides were greatly enhanced In electrorpray m a r spectra by addlng 10 mM sodium acetate or ammonium acetate to the sample solvent. Tandem mass spectrometrywas usedto determine prhnary structuralfeatures of the oilgoraccharides. Methoddogythat has been devdoped on branchedhlgh-mannom, hybrid, and complex carbohydrate standards was applied to a mlxture of ollgosaccharldes that were dlgested with N-glycanare from the glycoproteln, ovaibumin. The compodtlonand relativeabundance8of individual ollgoraccharldes obtalned from the electrorpray mass spectrum compare favorably to those obtained by anion-exchange chromatography/pulsed amperometric detectlon and by gel permeation chromatography of the oilgosaccharidesafter radiolabeilng the reduclng end of the carbohydrates. The olC gosaccharide content of ovalbumin was independently determined from the heterogeneity observed In the electroopray mass spectrm of the Intact 44kDa glycoprotein. C o m p a r h of the oligosaccharide compodtions determined before and after enzymatlc dlgestlonshows a mlectlve dlgestlon of highmannose and low molecular welght oligosaccharides by H giycanase.

INTRODUCTION Many proteins of therapeutic value are glycoproteins, including antibodies, hormones, growth factors, clotting factors, immunomodulators, and cell-surfacereceptors.' Frequently, the oligosaccharide contributes significantly to the biochemical and biophysicalproperties of a glycoprotein,such as to its half-life, its stability, its resistance to proteolysis, and ita biological specificity. For example, the oligosaccharides of the thrombolytic agent, tissue plasminogen activator (t-PA),play a direct role in shortening the biological half-life of the protein in serum as well as increasing or decreasing the enzymatic activity of the protein depending upon the oligosaccharide s t r u c t ~ r e . ~Likewise, *~ the in vivo activity of

* Author to whom correspondence should be addressed.

+ Current address: Schering Plough Research, 86 Orange Street,Bloomfield, NJ 07003. (1) Cumming D. A. Glycobiology 1991,1, 115. ( 2 ) Hotchkiss, A.; Refino, C. J.; Leonard, C. K.; O'Connor, J. V.; Crowley, C.; McCabe, J.; Tate, K.; Nakamura, G.; Powers, D.; Levinson, A.; Mohler, M.; Spellman, M. W. Thromb. Huemostusis 1988,60, 255. (3) Howard, S. C.; Wittwer, A.; Welply, J. K. Glycobiology 1991, I, 411.

0003-2700/92/0364-1440$03.00/0

erythropoietin, a drug being employed for treatment of anemia, is dramatically lessened by desialylation of its oligosaccharides, and recombinant forms of the protein containing highly-branched oligosaccharideshave greater in vivo activity than those containing oligosaccharides with less branching.* During preparation of Ceradase, a glucocerebrosidase used for treatment of Gaucher disease, the placentalderived aglucerase is treated with glycosidasesto modify its oligosaccharidesfrom complex types to those which terminate with mannose residues. The high-mannose oligosaccharides selectively target the glycoprotein to a macrophage, the cell where the activity is most critical, via an interaction between the high-mannose chains and a macrophage-specific cellsurface mannose receptor.5 Improvements in analytical methodologies for the determination of oligosaccharidestructure are needed to expedite studies of the biological functions of oligosaccharides. Currently, the characterization of oligosaccharide structures is often difficult and laborious because the supply of the oligosaccharide is usually limited, and their complexity and branching patterns make them difficult to fully characterize. Techniques for structure elucidation generally require derivatization and the availability of significant quantities. Analysis by treatment with glycosidases has proven to be a valuable technique for determining the sequence of oligosaccharides when this method is used in conjunction with sensitive separation techniques to evaluate alterations in the carbohydrate structure following enzymatic treatment.6 1H nuclear magnetic resonance (NMR) can provide complete structural information, including anomericity, linkage positions, sequence, and composition. However, this procedure is usually applicable only in cases where milligram quantities of material are available,' although there have been some reports of carbohydrate structure determination on smaller amounts of "plea8 Elucidation of the glycan structures that anchor coat proteins to the cell surface of parasites is a prime example of a biomedical application of this technique.9 Mass spectrometry has also proven valuable as an analytical tool for characterizing oligosaccharides and other carbohydrates.lOJ1 Fast atom bombardment (FAB) mass spectrometric characterization of both underivatized and derivatized oligosaccharides has provided the molecular weight of the (4) Takeuchi, M.; Kobata, A. Glycobiology 1991,1, 337. (5) Barton, N. W.; Furbish, F. S.; Murray, G. J.; Garfield, M.; Brady, R. 0. F'roc. Nutl Acad. Sci. U.S.A. 1990,87, 1913. (6) Yamashita, K.; Mizuochi, T.; Kobata, A. Analysis of Oligosaccharides by Gel Filtration. Methods Enzymol. 1982,83, 105. (7) Drabowski, J. (1989) T w o dimensional proton magnetic resonance spectroscopy. Methods Enzymol. 179, 122-156. (8) Nilsen, B. M.; Sletten, K.; Smestad Paulsen, B.; O'Neill, M.; van Halbeek, H. J. Biol. Chem. 1991, 266, 2660. (9) Ferguson, M. A.; Homans, S. W.; Dwek, R. A.; Rademacher, T. W. Science 1988, 239, 753. (10) Reinhold, V. N.; Carr, S. A. Muss Spectrom. Reu. 1983, 2, 153. (11)Dell, A. Adu. Carbohydr. Chem. Biochem. 1987, 45, 19. 0 1992 American Chemical Society

ANALYTICAL CHEMISTRY, VOL. 64, NO. 13, JULY 1, 1992

carbohydrate and yielded sequence information.12-14 Chemical derivatization of oligosaccharides by permethylation or peracetylation has been especially effective in enhancing the ion abundances of oligosaccharides and their fragments in FAB mass spectra.12 When FABMS is combined with exoglycosidase treatment and methylation analysis, complete structural characterization of carbohydrates is possible.1*17 Methylation analysis involvesgas chromatographic separation and mass analysis (GC/MS) of chemically-modified monosaccharides that are derived from methylation, hydrolysis, reduction, and acetylation of 0ligosaccharides.~8J9 The oligosaccharide composition and information about the linkages between individual monosaccharide residues is obtained by this technique. Both methylation analysis and FABMS generally require microgram quantities of oligosaccharides. Electrospray mass spectrometrym*21promises to complement other analytical methods for characterizing oligosaccharide structure by virtue of ita good sensitivity, its ability to detect underivatized carbohydrates and measure most of the components in a carbohydrate mixture, and ita ability to accurately measure the masses of glycoproteins and resolve oligosaccharide heterogeneity on the glycoprotein. This paper presents experimental conditions for electrospray and tandem mass spectrometric characterization of complex, underivatized N-linked oligosaccharides. The N-linked structures have a common core comprised of Manz-Man-GlcNAc-GlcNAc, which is extended with branched chains containingadditional mannose, N-acetylglucosamine, galactose, and sialic acid residues. In addition, data are presented on the analysis of the heterogenous mixture of N-linked oligosaccharides obtained from ovalbumin, a glycoprotein whose oligosaccharides have been extensively characterized by several techniques. The structural data obtained from electrospray mass analysis of N-glycanase-released oligosaccharides are compared to those generated by enzymatic sequencing and to those obtained from the electrospray mass spectrum of intact ovalbumin before the oligosaccharides are removed.

EXPERIMENTAL SECTION Materials. Carbohydrate standards were purchased from Dionex Corp. (Sunnyvale, CA) in a kit that contained 15 pg each of high-mannose and complex oligosaccharides. Each of the standards was judged >99% pure by anion-exchange chromatography. The lyophilized sugars were diluted to 250 pL with H2OMeOH (41,v:v) containingeither lOmMammoniumacetate, 10mM sodium acetate, or 2 % formicacid before characterization by electrospray mass spectrometry. Ovalbumin was purchased from SigmaChemicalCo. (St. Louis,MO) as alyophilized powder and diluted to a concentration of 100 pmol/pL in H20:AcN (2:1, v:v) containing 2 % formic acid. N-Glycanase, which was used for the enzymatic digestion of oligosaccharidesfrom ovalbumin, was purchased from Genzyme (Cambridge, MA). Purified exoglycosidases, Jack bean a-mannosidase and Jack bean &hexosaminidase, were kindly supplied by the Glycobiology Unit at (12) Dell, A.; Egge, H.; Von Nicolai, H.; Strecker, G. Carbohydr. Res. 1983, 15, 41. (13) Kamerling, J. P.; Heerman, W.; Vliegenthart, J. F. G.; Green, B. N.; Lewis, I. A. S.; Strecker, G.; Spik, G. Biomed. Mass Spectrom. 1983, 10, 420. (14) Carr, S. A,; Reinhold, V. N.; Green, B. N.; Hass, J. R. Biomed. Mass Spectrom. 1985, 12, 288. (15) Dell, A.; York, W. S.; McNeil, M.; Darvill, A. G.; Albersheim, P. Carbohydr. Res. 1983, 117, 185. (16) Fukuda, M.; Dell, A.; Fukuda, M. N. J.Biol. Chem. 1984,8,4782. (17) Spooncer, E.; Fukuda, M.; Klock, J. C.; Oates, J. E.; Dell, A. J. Biol. Chem. 1984,259,4792. (18) Hakomori, S. J. Biochem. (Tokyo) 1964,55, 205. (19) Stellner, K.; Saito, H.; Hakomori, S. Arch. Biochem. Biophys. 1973,155,464. (20) Bruins, A. P.; Covey, T. R.; Henion, J. D. Anal. Chem. 1987,59, 2642. (21! Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Scrence 1989, 246, 64.

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OxfordUniversity. [3H]NaBH4(10Ci/mmol)was obtained from New England Nuclear (Boston, MA). Bio-Gel P-4 (-400 mesh) was obtained from Bio-Rad (Richmond, CA). Enzymatic Digestion of Ovalbumin Oligosaccharides. Ten milligrams of ovalbumin was dissolved in 200 pL of 200 mM sodium phosphate buffer containing 1% 2-mercaptoethanol and 1%sodium dodecyl sulfate (SDS). The sample was heated to boiling for 5 min and diluted to 1 mL with sodium phosphate buffer containing 0.6% NP-40, a nonionic detergent. Twenty milliunits of N-glycanase then was added to this mixture, and the enzymatic reaction was allowed to proceed for 12 h at 37 O C . Afterward, an additional 20 munits of N-glycanase was added and the mixture was incubated for another 24 h. The sample mixture then was diluted to 70% ethanol and incubated for 2 h at 4 "C. Precipitated protein was removed by centrifugation. Nonionic detergent was removed from the supernatant by application on two C-18 Sep-Pak cartridges (Waters Chromatography Division, Milford, MA) in succession, and salts and ionic detergent were removed by application on Dowex AGdOW X-12 (H+form) and QAE-Sephadexcolumns in succesion. The deionized sample solution was evaporated under vacuum and redissolved in water. Twenty percent was utilized for electrospray mass analysis. EndoglycosidaseH digestion of ovalbumin was accomplished through treatment of the protein (1mg dissolved in 200 mL of a 20 mM sodium phosphate solution at pH 6.0) with 2 munits of endoglycosidaseH (Boehringer Mannheim) for 36 h at 37 OC. After digestion, the reaction mixture was applied to a Sephadex G-10 column that was equilibrated with 100 mM ammonium acetate. The void volume was collected,lyophilized,and analyzed by electrospray mass spectrometry. Chromatographic Characterization of Ovalbumin Oligosaccharides. A portion of the ovalbumin oligosaccharide mixture was applied to a 4.6- X 250-mm Dionex Carbo-Pac 1 anion-exchangecolumn, and carbohydrates were eluted isocratically at 1mL/min in 100 mM NaOH containing 25 mM NaOAc for 15 min followed by a gradient containing increasing levels of NaOAc from 25 to 100 mM NaOAc from 15 to 40 min. Oligosaccharideswere detected by pulsed amperometry. Individual peaks eluting from the anion-exchange column were collected, applied to a Dowex AG-50 X-12 (H+) column, evaporated to dryness, and radiolabeled by treatment with [3H]NaBHr,using standard procedures.6 The radiolabeled oligosaccharideswere separated from reaction byproducta by descending paper chromatography developed in BuOH:EtOH:H20 (4:1:1, v:v:v), and salts were removed by sequential application to columns containing Chelex-100 (Na+ form), Dowex AG-50 X-12 (H+form), Dowex AG3 (OAc- form),and QAE Sephadex. Oligosaccharides were eluted in water and evaporated to dryness. Filtered samples were applied to a gel permeation column of Bio-Gel P-4, (1.5 X lWcm, -400 mesh) and developed in water at 55 O C . A mixture of glucose polymers, obtained by partial acid hydrolysis of dextran, was co-injectedwith the radiolabeled sample as an internal standard. Radioactive sugars and coinjected glucose polymers were detected by autoradiography and refractive index changes, respectively. The radioactive sugars were fractionated, and their elution positions relative to that of glucose polymers were measured. The individual fractions from the P-4 column then were treated separately with purified exoglycosidases and reapplied to the same columnfor determination of changes in their elution position relative to the internal standards. HexNAc and Hex compositions were assigned for ovalbumin oligosaccharides based upon the results of the enzyme treatments. These values were compared to those observed by electrospray mass spectrometry. Electrospray Mass Spectrometry. A Sciex TAGA 6000E triple-quadrupole mass spectrometer equipped with an atmosphericpressure ion sourcewas used to samplepositive or negative ions produced from a pneumatically-assisted electrospray (ionspray) interface.20 Sample mixtures of oligosaccharides were introduced continuously through the ion spray interface at a rate of 2 pL/min. Positive or negative gas-phase ions that were created during nebulization and desolvation of analyte solutions were sampled through a 100-pm-i.d. conical orifice into the vacuum chamber for mass analysis. Pneumatic assistance was provided by passing approximately 1.5 L/m of nitrogen through

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a concentriccapillary that terminated approximately1mm before the end of the sprayer capillary. The atmospheric side of the conical orifice was bathed with a curtain of high-purity, dry nitrogen gas,which prevented contamination of the vacuum with solvent vapors and atmospheric gases and helped desolvate ions formed during nebulization of sample solutions. Ions entering the mass spectrometer were focused with an rf-only quadrupole (denoted as QO to differentiate it from the first quadrupole 81) into the mass analyzer and separated from neutral nitrogen molecules, which were frozen onto cryogenically-cooledsurfaces (15-20 K) that surround the first and second quadrupoles. In this first region of the mass spectrometer, sample ions encounter an area of declining pressure as neutral molecules are pumped away. Increasing the potential difference between the conical orifice and QOin this region causes greater desolvation and/or fragmentation of analytes. In this study, the potential difference was maintained at approximately 30 V, which resulted in full desolvationof analyte ions without dissociatingthe covalentbonds of the analyte. Fragmentation of protonated and ammoniated sample compounds could be accomplished by increasing this potential difference to 50-80 V. In this study, (M + 2NHd2+ were dissociated to (M + 2H)2+without significant oligosaccharide fragmentation by maintaining a voltage difference of 40 V in this region of the mass spectrometer. A working pressure of 2 X 10-5 Torr was maintained in the analyzer chamber during routine operation of the instrument. Mass analysis of sample ions was accomplished by scanning the first quadrupole (81) in 0.1-1.1increments from 200 to 1400 u in approximately 10 s, and passing mass-selected ions through the second (Q2) and third (83)quadrupoles operated in the rfonly mode to the multiplier, which was operated in the pulsecountingmode. Some of the electrospray mass spectra presented in this paper were acquired on an updated Sciex TAGA 6000E mass spectrometer that has a mass range of 2400 u. Operating conditions of the updated mass spectrometer were the same as those described above except that scan times from 200 to 2400 u were lengthened to 20 s. Mass spectra of oligosaccharideswere averaged over 5 scans for all results presented in this study. The mass spectrum of ovalbuminwas acquired by scanning from 1000 to 2400 u in 10 s. Forty scans were averaged to generate the signal/noise and resolution necessary to resolve oligosaccharide heterogeneity for this large glycoprotein. Positive-product-ion MS/MS spectra of oligosaccharideions were acquired by passing the (M + 2H)2+,(M + Na)+,or (M + 2Na)2+of individual oligosaccharidecompounds that had been mass-selected with the first quadrupole into the second quadrupole where they were dissociated by collision with ultrapure argon gas. Fragment ions generated by these collisionsthen were mass-analyzedby the third quadrupole, and detected. The third quadrupole was scanned in 0.1-11 increments from 10 to 1400 u in approximately 10 s, and 20 scans were averaged for each MSI MS spectrum. The argon target gas density was maintained at 2.3 X 1014 atom/cm2for this study, and collision energies were chosen by adjusting the dc voltage offset of the collision quadrupole. For this study collision energies of 50-100 eV (collision energy measured as the voltage difference between QOand Q2) with respect to the laboratory reference were used. The first and third quadrupoles were adjusted to obtain unit mass resolution at 80% peak height for mass spectra and MS/MS product ion spectra of singly-charged oligosaccharide standards. The ovalbumin mass spectrum was obtained with the resolution adjusted to unit mass resolution at 20% peak height.

RESULTS AND DISCUSSION Electrospray Mass Spectrometric Characterization of Oligosaccharide Standards. Eleven carbohydrate standards were characterized by electrospray and tandem mass spectrometry. These standards include high-mannose and complex oligosaccharides that contain various branching patterns and monomeric sugar units. All of the standards were isolated from the asparagine side chain of various glycoproteins and are representative of the type of N-linked oligosaccharides that commonly are encountered in biological systems. This study does not include characterization of 0-

linked oligosaccharides, which have been characterized by other methods and reported e l s e ~ h e r e ?but ~ ' ~the ~ methodology developed in this study should be equally applicable to these sugars, too. Oligosaccharides that did not contain sialic acid monomers were detected with the best sensitivity in the positive-ion detection mode in this study. Positive-ionelectrospraymass spectra of oligosaccharide standards were obtained by dissolving approximately 15 pg of each carbohydrate in a 250p L watermethanol (7:3,v:v) solution that contained either 10 mM ammonium acetate or 10 mM sodium acetate. Protonated oligosaccharides were not generated in appreciable abundance in this study when the carbohydrate solutions were acidified, so positive-ion electrospray mass spectra were acquired after addition of ammonium acetate or sodium acetate to the carbohydrate solution. Addition of these buffers resulted in the production of ammoniated or natriated oligosaccharides, respectively, by the electrospray process. Addition of salts, including ammonium salts, to sample matrices has also been found to significantly enhance the positive-ion sensitivity for carbohydrates analyzed by FABMS.24 Abundant protonated oligosaccharide ion signals were only generated in electrospray mass spectra by subjecting ammoniated oligosaccharides to collision-induced dissociation (CID) in the free-jet-expansion region of the API interface (see Experimental Section for details), which resulted in fragmentation of this ion by loss of neutral ammonia to form the protonated molecule. Similar results have been reported for the electrospray mass spectrometric characterization of acylglycerol compounds.25 In the present study it was found that adjustment of the CID potential difference to different values helped differentiate between different charge states of the oligosaccharide ions and helped to differentiate between the chargedonating species and, therefore, was important for determining molecular weights of the oligosaccharides. Figure 1exhibits the effect of this CID process on the molecular adduct ion region of an oligosaccharide. When the CID potential differencewas adjusted to 10 V, the (M + 2NH4)2+at m / z 839 was the most abundant ion in this region of the mass spectrum. When the CID potential difference was increased to 40 V, protonated molecule signals became more abundant as neutral ammonia was expelled from (M + 2NH4)2+. If the CID energy was further increased to 70 V, protonated and ammoniated oligosaccharides fragmented to lower-mass ions and the more stable sodium and potassium adduct ions (vide infra) increased in relative abundance to the ammoniated and protonated adduct ions. The mass spectra shown in Figure 1also illustrate how the charge state of oligosaccharide ions can be deduced from the addition of different ionizing species to the oligosaccharides. Mass-to-charge differences of half the mass difference of a proton and an ammonium or sodium ion in the mass spectra of Figure 1 indicate the doubly-charged oligosaccharide is observed in this region of the mass spectra. AU of the oligosaccharides that contain sialic acid monomers were detected with the best sensitivity in the negative-ion mode, presumably because the acidic carboxylic acid moiety a t the 1-position of sialic acid readily deprotonates under electrospray conditions to yield negatively-charged carbohydrate ions. Negative-ion electrospray mass spectra of sialylated oligosaccharides were obtained after dissolving the (22) Hounsell, E. F.; Wood, E.; Feizi, T.; Fukuda, M.; Powell, M. E.; Hakomori, S.Curbohydr. Res. 1981,90, 283. (23) Fukuda, M.; Carlsson, S.R.; Klock, J. C.; Dell, A. J. Biol. Chem. 1986,261, 12796. (24) Dell, A.; Oaks, J. E.; Morris, H. R.; Egge, H. Int. J. Muss Spectrom. Ion Phys. 1983,46, 415. (25) Duffin, K. L.; Henion, J. D.; Shieh, J. J. Anal. Chem. 1991, 63, 1781.

ANALYTICAL CHEMISTRY, VOL. 84, NO. 13, JULY 1, 1992 10 V Potential

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the conditions chosen for the analysis, the maximum number of negative charges that are present on the oligosaccharide corresponds to the number of sialic acid residues present on the compound. Each of the mass spectra contain the deprotonated oligosaccharide as the most abundant ion and also contain a less abundant adduct ion that results from addition of acetate. The acetate adduct ion is helpful for determining the charge state of the oligosaccharide because the mass-to-charge difference between it and the deprotonated oligosaccharide equals the combined mass of acetate and a proton divided by the number of charges. In Figure 2A the mass difference between the deprotonated oligosaccharide and ita acetate adduct ion is 30 u, which indicates the charge state of the oligosaccharideions is -2. Using the same reasoning, the oligosaccharide ions in Figure 2B correspond to the -3 charge state. With a quadupole mass analyzer of greater mass range (2400 u), lower charge states of the oligosaccharides could also be observed, which helped in determining the true charge state of the observed ions and, therefore, helped in determining the molecular weight of the carbohydrate. Tandem M a s s S p e c t r o m e t r i c C h a r a c t e r i z a t i o n of Oligosaccharide S t a n d a r d s . Molecular adduct ions of the branched oligosaccharidesthat were formed by electrospray were subjected to collision-induceddissociation in the second quadrupoleregion of the triple quadrupole mass spectrometer. Fragment ions that were formed by this collision process then were mass analyzed to determine primary structural features of the carbohydrate compounds. In general, the doublycharged or triply-charged oligosaccharidewas chosen for MS/ MS characterization because the singly-charged compounds had mlz values that were greater than the mass range of the mass analyzer. Collision-induceddissociation of sodiated oligosaccharides required high collision energies, and MS/MS spectra of sodiated oligosaccharides lacked significant fragment ion abundances. Even at high collision energies (100eV), product ion abundances were low relative to the parent ion, which resulted in MSIMS spectra with poor signal-to-noise. In fact, (M + 2Na)2+ ions showed greater susceptibility to dissociation through loss of Na+ to form (M + Na)+ than to dissociation through fragmentation of the carbohydrate bonds. Increased primary oligosaccharide information was available when protonated oligosaccharides were dissociated to generate MSIMS product ion spectra. Protonated oligosaccharides were generated by dissociatingammoniated oligosaccharides in the source region of the mass spectrometer (vide intra) and were preferred to ammoniated oligosaccharides for collision-induced dissociation studies to avoid any confusion in interpretation of MSIMS spectra. Figure 3, panels A and B, shows the MSIMS spectra of the (M 2H)2+for twodifferent oligosaccharideswhose structures are included in the figure. The most abundant product ion in each MS/MS spectrum results from cleavage of the glycosidic bond adjacent to an N-acetylglucosamine with charge retention by the nonreducing end of the sugar. In Figure 3A this cleavage mechanism yields the most abundant product ion at mlz 204, which corresponds to the oxonium ion of a -GlcNAc residue, and in Figure 3B it yields the most abundant product ion at mlz 366, which corresponds to the oxonium ion of a -Gal-GlcNAc residue. Preference for this cleavage mechanism in carbohydrates has been observed in FAB mass spectra and MSIMS spectra of permethylated and underivatized sugars.14~26 However, in the present case doublyprotonated oligosaccharides are fragmented by the CID process, and positive charge retention by the nonreducing

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carbohydrate in watermethanol (7:3, v:v), which contained 10 mM ammonium acetate (measured pH was 6.8). Under these conditions, the ion current of these oligosaccharides in the positive-ion mode was either very low or was not detected. The buffer solution used for negative-ion mass analysis of oligosaccharides was intentionally chosen to be the same as that used for positive-ion mass analysis so that ion detection could be “toggled” between the two modes for solutions of unknown oligosaccharides that might not be available in sufficient quantity for detailed sample preparation. Also, the same sample concentrations and flow rates that were used for positive-ion analysis were used for negative-ion analysis. Figure 2, panels A and B, shows the negative-ion electrospray mass spectra of two complex oligosaccharides. Under

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(26) Egge, H.; Dell, A.; von Nicolai, H.Arch. Biochem. Biophys. 1983, 224, 235.

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Flgure 4. Posltive-Ion MS/MS spectrumof the (M + 2H)2+ of a complex biantennary oligosaccharide. The Ion abundanceshave been multiplied 8X to clearly show low abundance product ions. G denotes Kacetylhexosamine residues, h denotes hexose residues, and f denotes fucose residues. The carbohydratestructure is drawn to reproduce the structure supplied wlth this commercial oligosaccharide standard. end of the compound must also be accompanied by a positive charge remaining on the reducing-end fragment. It is interesting, therefore, that the majority of ion current resides in these nonreducing-end fragments. Indeed, for all protonated oligosaccharidesthat were characterized by MS/MS in this study, the most abundant product ions in the MSIMS spectra resulted from dissociation by this fragmentation pathway. In low relative abundances in the MS/MS spectra of protonated oligosaccharides are fragment ions that result from dissociation of other glycosidic bonds of the oligosaccharide. Figures 4 and 5 show the MSIMS spectra of the (M + 2H)2+ of a biantennary oligosaccharide that contains a core fucose residue and a complex tetraantennary oligosaccharide. The ion abundances in these MSIMS spectra have been multiplied by 8x. Although the fragment ions in the MSIMS spectra are numerous, many are of only 1-2 % abundance relative to the most abundant product ions. Nevertheless, some sequence information can be deduced from the product ions that are present (see labeled fragment ions in Figures 4 and 5). However, the total unambiguous sequence of oligosaccharides could not be obtained from these MSIMS spectra

Figure 6. Positive-ion electrospray mass spectrum of an ollgosaccharide (M 2H)2+. Proposed product ion compositions are labeled. F denotes fucose, G denotes Nacetylhexosamine,and h denotes hexose residues. The carbohydrate structure Is drawn to reproduce the structure supplied wlth this commercial oligosaccharide standard.

+

because fragment ions result from both the reducing and nonreducing termini of the carbohydrate. Derivatization of oligosaccharides to yield fragment ions that originate only from the nonreducing terminus of oligosaccharideshas been useful in obtaining unambiguous sequence infromation from FAB mass spectra," and this procedure might be useful for obtaining unambiguous sequence information in the MS1 MS spectra of multiply-charged oligosaccharides that are generated by electrospray. When HexNAc residues were not present near the nonreducing termini of the oligosaccharides, more uniform abundances of product ions were observed in the MS/MS spectra. Figure 6 shows the MS/MS spectrum of an oligosaccharide (M + 2H)2+that exhibits this effect. Product ions that result from individual fucose, mannose, and N-acetylglucosamine residues are present at mlz 147, 163, and 204, respectively. Other product ions in the mass spectrum result from cleavage of glycosidic bonds, and their proposed compositions are labeled in the figure. The ion at mlz 470 probably results from cleavage of the mannose-N-acetylglucosaminebond to form an ion containing two GlcNAc and one fucose unit. One of the GlcNAc units then loses the N-acetyl group to form the ion at mlz 470. Alternatively, the commercial standard of


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d z

Id?.

Flgure 7. Positive-Ion eiectrospray mass spectrum of ovalbumin, a glycoprotein with one Ngiycosylationsite. Within each charge state of the glycoproteln at least slx different molecular weight giycoforms (labeled)that result from heterogenekyof oligosaccharidecomposition can be distinguished. Ovalbumin (M 24H)24+and (M 25H)25+are pictured in the flgure inset.

+

+

this oligosaccharide could contain an isomer that has fucose attached to mannose, but this possibility is improbable because the standard is chromatographicallypure and because fucose attachment to mannose is not known.

Characterization of Oligosaccharides Derived from Ovalbumin. The oligosaccharides of ovalbumin have been structurally characterized by several techniques, and there are numerous reports in the literature on their structures.27-32 Although ovalbumin has only a single glycosylation site, it contains 20 or more structurally different oligosaccharides, consisting of those that terminate in mannose residues only (high-mannosetypes) and those that terminate in mannose and N-acetylglucosamine (hybrid types1.31 To test the utility of electrospray mass spectrometry as a method for structure elucidation of oligosaccharides contained on glycoproteins, intact ovalbumin and free oligosaccharides derived from the glycoprotein were mass analyzed using this technique. The electrospray mass spectrum of ovalbumin contains multiple charge states of the glycoprotein, which result from attachment of differing numbers of protons to the ovalbumin molecule (Figure 7). Within each of these charge states, multiple forms of the glycoprotein are clearly resolved, and average molecular weights are assigned to each of these forms based on published equations.33 The inset of Figure 7 shows the (M + 24H)24+and (M + 25HP5+ region of the electrospray mass spectrum of ovalbumin with molecular weight values assigned to each of the individual glycoforms. Mass differences within 2 u of 162 and 203 u are apparent in this inset, which result from mass differences of carbohydrate hexose (mannose and galactose) units and N-acetylhexosamine (N-acetylglucosamine) units, respectively. The average molecular weight of the polypeptide portion of ovalbumin excluding posttranslational modifications is (27)Tai, T.; Yamashita, K.; Ogata-Arakawa,M.; Koide, N.; Muramatau, T.;Iwashita, S.; Inoue, Y.; Kobata, A. J.Biol. Chem. 1975,250, 8569. (28)Tai, T.; Yamashita, K.; Ito, S.; Kobata, A. J. Biol. Chem. 1977, 252,6687. (29)Yamashita, K.; Tachibana, Y.; Kobata, A. J. Biol. Chem. 1978, 253,3862. (30)Chen, L.-M.; Yet, M.-G.; Shao, M . 4 . FASEB J. 1988,2, 2819. (31)Yet, M.-G.;Chin, C. C. Q.; Wold, F. J.Biol.Chem. 1988,263,111. (32)Maley, F.; Trimble, R. B.; Tarentino, A. L.; Plummer, T. H., Jr. Anal. Biochem. 1989,180, 195-204. (33)Covey, T. R.;Bonner, R. F.; Shushan, B. I.; Henion, J. D. Rapid Commun. Mass Spectrom. 1988,2,249.

Flgure 8. Posltlveioneiectrospray mass spectrum of ovalbuminafter enzymatic digestion of the N-linked carbohydrates with endoglycosidase H. EndoglycosldaseH cleaves after the first GlcNAc unit, so the observed molecular weight of 43 073 u includes the resldue mass of GlcNAc (203 u).

known from translation of the mRNA34and DNA35sequences to be 42 750 u. Posttranslational modifications that have been reported include acetylation of the N-terminus, phosphorylation of Ser-68, and formation of a disulfide bond that links cysteine residues 73 and 120.36 These modifications account for an additional 119 u, which when added to the previous figure of 42 750 u yields a calculated average molecular weight of 42 869 u for ovalbumin, excluding the mass of the N-linked carbohydrate portion of the glycoprotein. This molecular weight was confirmed by electrospray mass analysis of ovalbumin after the N-linked carbohydrate was cleaved from the protein with endoglycosidase H. Figure 8 shows the mass spectrum of the digested protein, which yielded an average molecular weight of 43 073 u. Endoglycosidase H cleaves N-linked oligosaccharides between the two reducing-end N-acetylglucosamineunits, so the observed molecular weight includes the mass of a HexNAc residue (203 u). After subtraction of the HexNAc residue mass, the calculated averagemolecular weight of the polypeptide portion of ovalbumin is 42 870 u. The carbohydrate digestion was also performed with N-glycanase,but the polypeptide portion of ovalbumin precipitated from solution and could not be resolubilized for mass analysis. The heterogeneous ion signals of low relative abundance in the mass spectrum of Figure 8 result from incomplete digestion of ovalbumin by endoglycosidase H. Subtraction of the observed molecular weight of the polypeptide portion of ovalbumin (42 870 u) from the observed average molecular weights of the individual glycoforms of ovalbumin that are recoreded in the electrospray mass spectrum (Figure 7) yields the residue masses of the different oligosaccharidesthat are attached a t the N-glycosylationsite of the glycoprotein. Addition of 18 u (from water loss during formation of the asparagine-carbohydrate bond) to the oligosaccharide residue masses then yields the oligosaccharide molecular weights. In Figure 7 average ovalbumin molecular weights of 44 087 f 1, 44249 f 1, 44 492 f 3, 44 655 f 1, 44 695 f 3, and 44 857 f 5 translate into oligosaccharide molecular weights of 1235, 1397, 1640, 1803, 1845, and 2007, (34)McReynolds, L.; OMalley, B. W.; Niabet, A. D.; Fothergill, J. E.; Givol, D.; Fields, S.; Robertson, M.; Brownlee, G. G. Nature 1978,273, 722. .

(35)Woo, S. L.C.; Beattie, W. G.; Catterall, J. F.; Dugaiczyk, A.; Staden, R.; Brownlee, G. G.; OMalley, B. W. Biochemistry 1981,20,6437. (36)Thompson, E.0.P.; Fischer, W. K. A u t . J.Biol. Sci. 1978,31, 433.

1446


Table 1. Results of Figures 7 and 9 Compared to Show Molecular Weights (Ions) That Yield the Same Oligosaccharide Compositionsa

104

MW (Figure 7), u

AMW (Figure 7 ) , 4

carbohydrate

mlz (Figure 9), u

44087 44249 44492 44655 44695 44857

1234 1396 1639 1802 1844 2006

HexsHexNAcz He~HexNAcz HexsHexNAc4 He~HexNAc4 HexsHexNAc5 He-HexNAc5

1258 1420 1664 (843) xxx (946) xxx

The observed ions from Figure 9 are (M + Na)+ and (M + 2Na)2+ (listed in parentheses). The denotion xxx indicates that these oligosaccharides were not observed.

750

1000

1210

1500

1750

2000

m z

Flgure Q. Posltlve-ion electrospray mass spectrum of the oligosaccharides that were digested from ovalbumin wlth Kglycanase. The (M Na)+ and (M 2Na)*+ of high-mannose and complex oligosaccharides are more easily distinguished after removal from the asparagine side chain of ovalbumin (compare to Figure 7).

+

+

which are within 2 u of the correct mass of oligosaccharides that consist of HexsHexNAcz, Hex6HexNAcz,Hex~HexNAc~, HexsHexNAcr, Hex5HexNAc5, and Hex6HexNAc5, respectively. For validation of the oligosaccharide content of ovalbumin, the carbohydrate portion of ovalbuminwas enzymatically cleaved from the glycoprotein with N-glycanase and characterized by electrospray mass spectrometry, anion-exchange chromatography, and enzymatic methods coupled with gel permeation chromatography. Molecular weights of the N glycanase-released oligosaccharides were directly obtained by electrospray mass spectrometry without prior separation of the individual oligosaccharides in the enzymaticallydigested mixture. Methodology that was developed on oligosaccharide standards and presented earlier in this paper was also used to characterize this mixture. However, it was unnecessary to add sodium or ammonium to the sample solution for ionization of these oligosaccharides because residual sodium from the enzymatic reaction was already present. The ability of electrospray mass spectrometry to measure most of the components of a mixture from the same compound class without fragmenting the components, which would result in confusing mass spectra, has been documented for peptide37 and acylgly~erol~~ mixtures. The oligosaccharide mixture derived from ovalbumin also was characterized by electrospray mass spectrometry under conditions that did not produce fragmentation. It was hoped that the successful application of the technique toward other compound classes would also apply toward carbohydrate mixtures. Figure 9 shows the electrospray mass spectrum from the mixture of oligosaccharidesthat were enzymatically-digested from ovalbumin. Because it is easier to resolve low molecular weight oligosaccharidesin an electrospray mass spectrum than it is to resolve the oligosaccharides attached to a high molecular weight multiply-charged protein, greater oligosaccharide heterogeneity is apparent in the mass spectrum of Figure 9 than can be resolved from the mass spectrum of Figure 7. The two most abundant ions at mlz 1258 and 1420 result from the (M + Na)+ of oligosaccharides having the compositions HexsHexNAcz and He&HexNAc2. Numerous other high-mannose and hybrid oligosaccharides (M + Na)+ and (M + 2NaI2+are also observed in the electrospray mass spectrum of Figure 9, and their compositions are deduced (37) Chowdhury, S. K.; Katta, V.; Chait, B. T . Biochem. Biophys. Res. Commun. 1990, 167, 686.

from their observed molecular weights (see labeled ions in Figure 9). A comparison of the oligosaccharide composition of ovalbumin that is obtained from the electrospray mass spectrum of intact ovalbumin to that obtained from the electrospray mass spectrum of N-glycanase-releasedoligosaccharidesfrom ovalbumin shows some similarities and several differences. Molecular weight comparisons that yield the same oligosaccharide composition appear in Table I. The two most abundant oligosaccharides observed in the enzymaticallydigested mixture (Figure 9), Hex5HexNAcz and He%HexNAcz,are also observed in high abundance as glycoforms of intact ovalbumin (Figure 7). The most abundant glycoform in the mass spectrum of Figure 7 contains an oligosaccharide with composition Hex5HexNAc4, which is observed in high relative abundance in the mass spectrum of the N glycanase digest as (M + Na)+ and (M + 2NaY+. However, two of the other abundant ovalbumin glycoforms, He&HexNAc4and HexsHexNAc5,that are observed in the mass spectrum of Figure 7 contain oligosaccharides that are not observed in appreciable abundance in the mass spectrum of the N-glycanase digest (Figure 9). These differences in relative abundance suggest that either electrospray mass spectrometry selectively detected certain oligosaccharidesor that N-glycanase selectively digested these types of oligosaccharides from ovalbumin. T o determine whether electrospray mass spectrometric analysis accurately determined the oligosaccharide content of the N-glycanasedigest, anion-exchangeand gel permeation chromat,ographywere used to independently characterize the same oligosaccharidemixture. A portion of the N-glycanase digest was applied to a Dionex pellicular anion-exchange Carbo-Pac 1 column for separation into individual components (Figure 10). Several peaks were observed by pulsed amperometric detection and peaks 4-8, the most abundant carbohydrate signals, were fractionated for additional characterization. Before subjecting the samples to enzymatic treatments, sugars contained in peaks 4-8 were radioactively tagged by reduction with [3HlNaBH4and applied together with nonradioactive isomaltose standards to a Bio-Gel P-4 gel permeation column. The elution positions of t h e radioactive oligosaccharides originating from fractions 4-8 were compared to those of the standard glucose polymers to establish an initial P-4 elution position for the major components that eluted from the gel permeation column. These positions have been shown to be proportional to the hydrodynamic volume of the eluting oligosaccharides.6 The major radioactive species that were recovered from the P-4 column were fractionated and treated with Jack bean a-mannosidase or Jack bean P-hexosaminidase and reapplied to the same column to determine the effect of the enzymatic treatment on the elution position. An example of the enzymatic analysis is shown for material contained in peak 5 of the anion-exchange separation. The


Table 11. Sequential Enzymatic Sequencing of Dionex Fractions 4-8 with Jack Bean @-Hexosaminidaseor a-Mannosidase Established Hex/HexNAc Composition for Each Sugar Based upon Their Elution Positions on Gel Permeation Column

2

500

7

0

20

10 Relative Retention ( m i n )

Flguro 10. Separation of ovalbumin derived, Kgiycanassreleased oilgosaccharides by high-performance anion-exchange chromatography. Detection was accomplished by pulsed amperometry.

I 0 9 8 7 6 5 4 I

x

E >

A

I

I

I

I

I

I

3

2

1

I

I

I

h

m .-0 TJ

m

a:

&--@‘,””(

‘

W

Relative Retention

10987 6 5 4 I

I

I

I

1447

I

I

I

3 I

1

B .TJ

m

a:

Relatlve Retention Flguro 11. Gel permeatlon chromatographs of the oligosaccharide isolated as fraction 5 by anion-exchange chromatography. The top chromatogram(A) was obtained before and the bottom(B)after enzyme treatment wkh Jack bean a-mannosidase.

initial P-4 elution position of the major radioactive material obtained from reduction of peak 5 is 8.9 glucose units (Figure 11A). Based upon values in the literature6 and upon the published structures of ovalbumin sugars, an oligosaccharide with elution of 8.9 is likely to be the high-mannose type, Man5GlcNAcp,having four outer a-linked mannose units and a ,!?-linkedMan-GlcNAc-GlcNAccore common to all N-linked oligosaccharide structures. To confirm the structure of the 8.9 oligosaccharide, the sugar was treated with a-mannosi-

fraction

initial P-4 GU

enzyme

new P-4 GU

4a

12.3

4b

10.2

&Hex a-Man a-Ma @-Hex

5 6

8.9 13.5

7 8

11.9 9.7 11.6

7.2 5.5 10.2 7.2 5.5 5.5 7.2 5.5 7.2 5.5 10.6 6.5 5.5

a-Mm a-Mm j3-Hex a-Mm j3-Hex

a-Mm a-Mm j3-Hex

a-Mm

composition HexsHexNAc5 HexsHexNAc, HexsHexNAcz HexsHexNAc5 HexsHexNAcb He~HexNAcz HexsHexNAcd

dase which reduced its elution position to 5.5 glucose units (Figure l l B ) , the established value for the a-mannosidase resistant core, Man/3-GlcNAc,!?-GlcNAc-OH.Peak 5 from the anion-exchange column therefore contains an oligosaccharide that yields one of the major sodiated ions observed in the electrospray mass spectrum of N-glycanase-releasedoligosaccharides, HexbHexNAcz. A summary of the enzymatic results and the entire P-4 analysis is shown in Table 11. The other major component, contained in peak 7 (Figure lo), was sensitive to a-mannosidase treatment, and its structure is deduced to be Man6GlcNAcp or HexsHexNAcz, another major species observed in the electrospray mass spectrum of the unfractionated material. The remaining oligosaccharidesderived from ovalbumin in peaks 4,6, and 8 were found to require treatment with /3-hexosaminidase and a-mannosidase to be reduced to the core elution position of 5.5 glucose units, consistent with their containing terminal N-acetylglucosamine or N-acetylglucosamine and mannose residues (Le., hybrid structures). All oligosaccharides listed in Table I1 are observed in the electrospray mass spectrum, and the relative abundances of the N-glycanase-released oligosaccharides that were determined by the different analytical methods were similar. The remaining oligosaccharides that are observed in the electrospray mass spectrum (Figure 9) but not in the gel permeation chromatogram were either not present as abundant radiolabeled species in anion-exchange peaks 4-8 or were not in one of these fractions that were analyzed on the gel permeation column. Mass spectrometry was not able to differentiate structural isomers of the carbohydrates, so chromatographic selectivity was necessary to more fully characterize the oligosaccharide mixture. Table I1 shows that several of the sugars eluting from the anion-exchange column had the same carbohydrate composition (and mass), and so mass spectrometry was unable to differentiate these compounds. The results of our study agree well with published reports of the oligosaccharidestructures of ovalbumin.30-31Fourteen of the reported carbohydrate compositions were detected by electrospray mass spectrometric characterization of the Nglycanasedigest. Only a reported structure of He~HexNAcs was not observed in our study. Two structures that were observedin this study, HexsHexNAcz and HeqHexNAs, were not reported in the earlier study. The reported abundances of ovalbumin oligosaccharides31 were also similar to those observed in this study. The two most abundant carbohydrates observed in the earlier study were HexSHexNAcz and HeQHexNAcp,respectively, which are the most abundant oligosaccharides observed in the electrospray mass spectrum of the N-glycanase digest of ovalbumin (Figure 9). Other reported structures yielded abundances that were not absolutely the

1448


same but that were reasonably close to abundances observed in this study.

CONCLUSIONS Complex, underivatizedOligosaccharidesthat were removed from the N-glycosylation site of glycoproteins were characterized by electrospray and tandem mass spectrometry. Oligosaccharides that contained sialic acid residues were detected with the best sensitivity in the negative-ion detection mode after deprotonation. Oligosaccharides that did not contain sialic acid were best detected in the positive-ion mode after formation of adduct ions with ammonium or sodium. Ammonium acetate or sodium acetate was dissolved in 10 mM concentrations into the carbohydrate solution to facilitate cation attachment. MSIMS characterization of these complex oligosaccharides yielded fragment ions that resulted from cleavages of glycosidic bonds. Cleavages adjacent to N-acetylhexosamine residues were a preferred mechanism, and product ions resulting from this dissociation dominated the MSIMS spectra. Product ions resulting from dissociation of other glycosidic bonds also were present in MSIMS spectra in high enough abundance to determine some of the oligosaccharide sequence. However, the methodologypresented in this paper did not allow differentiation of isomeric sugar units such as mannose and galactose and did not allow determination of linkage positions. Electrospray mass analysis of ovalbumin, a glycoprotein with one glycosylation site, allowed differentiation of six different oligosaccharide molecularweights that were attached at the N-glycosylation site. The compositions of these oligosaccharides were identified by their molecular weights as Hex5HexNAc2,He@exNAc2, Hex5HexNAcr,HexsHexNAcl, HexsHexNAc5, and He~HexNAc5.When the oligosaccharides were removed from the glycoprotein with N-glycanase, electrospray mass analysis of the digested mixture allowed determination of more than 15oligosaccharideswith different

molecular weights. Better mass resolution of low molecular weight oligosaccharides allowed determination of more glycoformsof ovalbumin than could be directly determined from the mass spectrum of the intact glycoprotein. Oligosaccharideabundances obtained from the mass spectrum of the intact glycoprotein were found to differ from those obtained from the mass spectrum of the N-glycanasereleased oligosaccharide mixture. Two other analytical methods, ion chromatographylpulsed amperometricdetection and gel permeation chromatography/radioactivedetection, yielded digested oligosaccharideabundances that were similar to those obtained by electrospray mass spectrometric characterization of the N-glycanase digest. This agreement suggests that the N-glycanase digestion of ovalbumin oligosaccharides was not complete. However, results from this study cannot rule out a possibility that electrospray mass spectrometry detected some glycoforms of the intact glycoprotein with better sensitivity, thereby skewingthe observed abundances of those glycoforms in the electrospray mass spectrum of intact ovalbumin. Chromatographic separation of the N-glycanase-released oligosaccharides allowed differentiation of isomeric sugars that could not be differentiated by mass spectrometry. Work is underway to perform on-line ion chromatographylmass spectrometry of the N-glycanase-releasedsugars, which allows coupling the specific detection capabilities of mass spectrometry with the ability to resolve isomeric sugars with ion chromatography.

ACKNOWLEDGMENT K.L.D. initiated the work in this study a t Cornel1University while a postdoctoral associate in Professor Henion’s laboratory and thanks the Monsanto Co. for financial support during his postdoctoral term.

RECEIVED for review December 3, 1991. Accepted March 30, 1992.

Characterization of N-linked oligosaccharides by electrospray and

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