Anal. Chem. 1996, 68, 4424-4430
A Coordinated High-Performance Liquid Chromatographic, Capillary Electrophoretic, and Mass Spectrometric Approach for the Analysis of Oligosaccharide Mixtures Derivatized with 2-Aminoacridone George Okafo,† Louise Burrow,† Steven A. Carr,*,‡ Gerald D. Roberts,‡ Walter Johnson,‡ and Patrick Camilleri*,§
SmithKline Beecham Pharmaceuticals, The Frythe, Welwyn, Herts AL6 9AR, U.K., SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, Pennsylvania 19406-0939, and SmithKline Beecham Pharmaceuticals, Coldharbour Road, Harlow, Essex CM12 5AD, U.K.
Glycans derivatized with 2-aminoacridone have been analyzed consecutively by reverse-phase high-performance liquid chromatography (HPLC) and micellar electrokinetic capillary chromatography (MECC). The 2-aminoacridone derivatizing agent used in the present study is highly hydrophobic and is well separated from the glycan derivatives in both separation techniques, ensuring that excess reagent does not interfere with the oligosaccharide analysis. The methodology outlined uses the high resolving power of capillary electrophoresis to determine the heterogeneity of samples after collection and preconcentration by HPLC. Collected glycan samples are submitted for mass spectrometric analysis to determine molecular weight. This methodology has been applied to linear oligosaccharides derived from dextran and to N-linked mannose-rich glycans from ribonuclease B. Oligosaccharides can play important and diverse roles in the biological function of the proteins to which they are attached.1-3 They can play a general role in protein folding and can influence the recognition processes between a glycoprotein and other molecules.4 Their presence and/or heterogeneity may alter the overall physicochemical properties of proteins, such as solubility and protease resistance. The extent of glycosylation in an enzyme can also modulate its activity.5 Rapid acquisition of information on glycan heterogeneity may also be crucial in the control of the batch-to-batch consistency of the glycosylation of recombinant therapeutic proteins. In recent years, a number of strategies have been developed for the preparation, fingerprinting, and sequencing of oligosaccharides using picomolar amounts of glycoprotein. Chromatographic (in particular, size-exclusion and ion-exchange)6,7 and freezone capillary electrophoretic8,9 separation methods have featured †
SmithKline Beecham Pharmaceuticals, Herts, U.K. SmithKline Beecham Pharmaceuticals, King of Prussia, PA. SmithKline Beecham Pharmaceuticals, Essex, U.K. (1) Cumming, D. A. Glycobiology 1991, 1, 115-130. (2) Parekh, R. B. Curr. Opin. Struct. Biol. 1991, 1, 750-754. (3) Kobata, A. Eur. J. Biochem. 1992, 209, 483-501. (4) Helenius, A. Mol. Biol. Cell 1994, 5, 253-260. (5) Dwek, R. A. Chem. Rev. 1996, 96, 683-720. (6) Lee, Y. C. Anal. Biochem. 1990, 189, 151-162. ‡ §
4424 Analytical Chemistry, Vol. 68, No. 24, December 15, 1996
prominently in these studies. In conjunction with spectroscopic and spectrometric techniques, the structures of complex glycan structures have been determined over a shorter period of time than previously possible.10,11 Recently, we reported12-15 on the use of 2-aminoacridone (AMAC) as a derivatizing fluorophore for the rapid and highresolution analysis of mixtures of glycans by micellar electrokinetic capillary chromatography (MECC). The methodology we developed has a major advantage in that excess derivatizing reagent does not interfere with the analysis, as it is trapped by the taurodeoxycholate micelles, resulting in a considerably longer migration time than those of the derivatized oligosaccharides. Using this technique, we were able to fingerprint several heterogeneous glycan pools. In some cases, preliminary identification of individual components was carried out either by comparison of migration times with those of commercially available standards or by relating capillary electrophoresis (CE) and size-exclusion data. Definite structural identification is, of course, not possible by using only separation methodology. Although collection of analytes after separation by CE is possible,16 difficulty is usually encountered when the isolation of pure substrates is attempted from highly resolved mixtures where peaks migrate very close to one another. Another major disadvantage of operating this technique in this mode is that the amounts collected are small and may not be enough to obtain sequencing information; this is (7) Bigge, J. C.; Patel, T. P.; Bruce, J. A.; Goulding, P. N.; Charles, S. M.; Parekh, R. B. Anal. Biochem. 1995, 230, 229-238 (8) Honda, S.; Makino, A.; Suzuki, S.; Kakehi, K. Anal. Biochem. 1990, 191, 228-234. (9) Chiesa, C.; Horvath, C. J. Chromatogr. 1993, 645, 337-352. (10) Takahashi, N.; Ishii, I.; Ishihara, N.; Mori, M.; Tejima, S.; Jeffries, R.; Endo, S.; Arata, Y. Biochemistry 1987, 26, 1137-1144. (11) Huddleston, M. J.; Bean, M. F.; Carr, S. A. Anal. Chem. 1993, 65, 877884. (12) Greenaway, M.; Okafo, G. N.; Camilleri, P.; Dhanak, D. J. Chem. Soc., Chem. Commun. 1994, 1691-1692. (13) Camilleri, P.; Harland, G. B.; Okafo, G. Anal. Biochem. 1995, 230, 115122. (14) Harland, G. B.; Okafo, G.; Matejchuk, P.; Sellick, G. E.; Chapman, G. E.; Camilleri, P. Electrophoresis 1996, 17, 406. (15) Okafo, G. N.; Burrow, L. M.; Neville, W.; Truneh, A.; Smith, R. A. G.; Camilleri, P. Anal. Biochem. 1996, 240, 68-74. (16) Muller, O.; Foret, F.; Karger, B. L. Anal. Chem. 1995, 67, 2974-2980. S0003-2700(96)00721-4 CCC: $12.00
© 1996 American Chemical Society
Scheme 1
especially true in the case of glycans, where sequencing may involve a series of enzyme reactions. In contrast to CE, high-performance liquid chromatography (HPLC) is suitable for the isolation of substrates in sufficient quantities for 1H NMR spectroscopy, mass spectrometry, and sequencing studies.10,11,17 The use of AMAC in the chromatographic preparation of labeled oligosaccharides for spectroscopic and spectrometric analysis has provided valuable information on glycan structures. However, it has been reported recently18 that, in the case of the derivatization of small oligosaccharides, excess 2-aminopyridine can interfere with the isolation of substrates free of contamination with this reagent and can be removed by cationexchange chromatography. In this study, we show that the hydrophobic nature of AMAC makes it most suitable for the analysis and preparation of derivatized oligosaccharides of a variety of sizes using reversephase HPLC. Lack of interference by excess reagent with the analysis and fraction collection of glycans makes this separation technique ideal to develop HPLC methodology in concert with capillary electrophoresis and mass spectrometry (electrospray and matrix-assisted laser desorption/ionization (MALDI)). Here we demonstrate the usefulness of complementary and rapid methods in the analysis of a mixture of linear polysaccharides from a dextran pool and the mannose-rich glycan pool from ribonuclease B. EXPERIMENTAL SECTION Chemicals. Sodium hydroxide, boric acid, and glacial acetic acid were obtained from BDH (Poole, U.K.). The sodium salt of taurodeoxycholic acid was >99% pure from Sigma (Gillingham, U.K.). Dimethyl sulfoxide (DMSO), trifluoroacetic acid (TFA), and sodium cyanoborohydride were purchased from Aldrich (Gillingham, U.K.). The derivatization reagent, 2-aminoacridone, was synthesised using a slight modification of the method described by Barnett et al.:19 Crude 2-aminoacridone prepared from 4-acetamidodiphenyl-2-carboxylic acid was recrystallized from ethyl acetate, and petroleum ether was added slowly to precipitate the product. (17) Suzuki-Sawada, J.;Umeda, Y.; Kondo, A.; Kato, I. Anal. Biochem. 1992, 207, 203-207. (18) Fan, J.; Huynh, L. H.; Lee, Y. C. Anal. Biochem. 1995, 232, 65-68. (19) Barnet, M. M.; Gillieson, A. H. C.; Kermack, W. O. J. Chem. Soc. 1934, 433-435.
All buffers were prepared using distilled, deionized water (MilliQ), and pH adjustments were performed by the addition of dilute sodium hydroxide. The partially hydrolyzed dextran ladder and the glycan mixture from ribonuclease B were purchased from Oxford GlycoSystems (Abingdon, U.K.). Derivatization with 2-Aminoacridone (AMAC). Carbohydrates were derivatized with 2-aminoacridone according to the procedure previously reported by Jackson.20 The derivatization is a “one-pot” reaction, and the reactions involved are shown in Scheme 1 for the preparation of a glucose-AMAC derivative. Typical experiments involved using a weight (approximately 1 µg) of dried lyophilized glycan mixture, to which was added a volume (10 µL) of 0.1 M AMAC solution (2 mg of the solid dissolved in a 3:17 (v/v) mixture of glacial acetic acid and DMSO, respectively) in a clean Eppendorf tube. The mixture was then manually agitated for 30 s before the addition of an aliquot (10 µL) of a freshly prepared 1 M aqueous solution of NaCNBH3 (7 mg of the solid dissolved in water). The reaction was again agitated manually and then incubated at 90 °C for 30 min in the dark. The reaction was then stopped by immersing the Eppendorf tube in a cardice bath at -20 °C. An aliquot of the AMAC solution was diluted and analyzed by MECC and reverse-phase HPLC. Reverse-Phase HPLC Conditions. Chromatographic separation was performed using a Perkin-Elmer Series 4 liquid chromatograph, which was fitted with a Waters µBondapak C18 column (3.9 mm × 300 mm) or a Symmetry column (4.6 mm × 250 mm). About 100 ng of the AMAC derivatives was injected onto the columns and detected by fluorescence using the PerkinElmer LS-4 fluorescence spectrometer (λex ) 442 nm and λem ) 520 nm) with a slit width of 10 nm. The mobile phase was comprised of 100 mM ammonium acetate, pH 6.69 (solvent A), and acetonitrile (solvent B). The column was eluted under gradient conditions (flow rate, 1.0 mL/min): step 1, linear gradient of 10% B to 25% B over 45 min; step 2, equilibration of the column at 10% B for 5 min. HPLC fractions were collected manually by observing appropriate signals on a chart recorder (Kipp and Zonen, U.K.). These were concentrated to near dryness using a GeneVac centrifuge (Ipswich, U.K.) and reconstituted to about 5 µL with water before submission for CE analysis and mass spectrometric measurements. (20) Jackson, P. Anal. Biochem. 1991, 196, 238-244.
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CE Separation Conditions. The CE system consisted of a Beckman P/ACE Model 2100 equipped with a laser-induced fluorescence (LIF) detector and temperature-controlled autosampler. The untreated fused silica capillary used was 57 cm in length (50 cm effective length) and 50 µm i.d. The separation buffer contained 300 mM boric acid and 80 mM taurodeoxycholic acid adjusted to pH 9.2 with dilute sodium hydroxide. A voltage of 25 kV (I ) 130 mA) was applied across the ends of the capillary, and detection of analytes was carried out using laser-induced fluorescence (helium-cadmium, λex ) 442 nm, λem ) 520 nm). The outside temperature of the capillary was maintained at 25 °C. Derivatized glycans (∼50 ng) were introduced at the anodic end of the capillary by a 3 s high-pressure injection of diluted sample solution. Mass Spectrometric Analysis. (i) Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS). Mass spectrometric analysis of the derivatized ribonuclease B glycans was carried out using a TofSpec SE mass spectrometer (Micromass, Manchester, U.K.) in the reflectron mode, whereas analysis of the dextran “ladder” was carried out with an instrument of the same make operated in the linear mode. In both cases, photon irradiation from a 337 nm pulsed nitrogen laser and 25 keV accelerating energy were used. The instrument was externally mass calibrated using the (M + Na)+ peak of 2,5-dihydroxybenzoic acid and the (M + H)+ peak of a peptide of known Mr. A matrix solution was prepared by dissolving 7.5 mg of 2,5dihydroxybenzoic acid + 2.5 mg of 1-hydroxyisoquinoline (ratio of 3:1 w/w) in 1 mL of acetonitrile/water (1:1 v/v). Lyophilized samples of AMAC-glycans collected from HPLC were reconstituted in 15 (fractions 1 and 2) or 25 µL (fraction 3) of 20% acetonitrile in water. Portions of 0.5 µL each of sample and matrix were spotted on a stainless steel target, and the solvent was quickly evaporated with a stream of argon. Derivatized glycans were detected as the (M + Na)+ and (M + K)+ ions. (ii) Nanoliter Flow Electrospray MS. Electrospray (ES) mass spectra obtained at the nanoliter per minute flow rate (nanospray) were recorded on a Perkin-Elmer Sciex API-III triplequadrupole mass spectrometer (Thornhill, ON, Canada). This instrument is fitted with an articulated nanospray interface, developed by Wilm and Mann21,22 and built at the European Molecular Biology Laboratory in Heidelberg, Germany. The procedures for sample introduction and data acquisition are described in detail in ref 23. RESULTS AND DISCUSSION Separation of AMAC Derivatives by MECC and RP-HPLC Collection of Labeled Oligosaccharides. Dextran is a polysaccharide containing a backbone of D-glucose units linked R-1,6 to one another. Incomplete hydrolysis of this carbohydrate leads to a mixture made up of glucose and a number of poly-D-glucose units of different lengths. Recently, we reported13 the MECC analysis of an AMAC-derivatized carbohydrate pool from dextran, where we were able to obtain a high-resolution separation of about 22 components. The migration behavior of these linear oligosaccharides, composed of (6-GluR1-) repeating units, was found to be related to size: solutes with the largest hydrodynamic volume (21) Wilm, M.; Mann, M. Int. J. Mass Spectrom. Ion Processes 1994, 136, 167180. (22) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8. (23) Carr, S. A.; Huddleston, M. J.; Annan, R. S. Anal. Biochem. 1996, 239, 180192.
4426 Analytical Chemistry, Vol. 68, No. 24, December 15, 1996
Figure 1. Hydrolyzed dextran ladder analyzed by reverse-phase HPLC using (a) µBondapak and (b) Symmetry columns. The AMAC carbohydrate derivatives elute before excess AMAC, denoted by the largest signal toward the end of the chromatograms. The numbers shown relate signals to the number of glucose units in the derivatized carbohydrate.
migrated first and glucose last. This order of migration is the same as that obtained using size-exclusion chromatography, though with much less resolution. The use of an MECC mode allowed the efficient trapping of excess AMAC by the taurodeoxycholate micelles, ensuring no interference with the analysis of the oligosaccharide mixture. The preferential distribution of the free AMAC onto the micelle wall is due to its relatively high lipophilicity, compared to those of the AMAC-derivatized carbohydrates. We have now made use of the hydrophobic nature of AMAC to achieve separation of the components of a dextran ladder by reverse-phase HPLC. Results are shown in Figure 1, which compares data obtained using two types of reverse-phase supports: a µBondapak column (10 µm particle size) and a Symmetry column (5 µm particle size). Particle size has a profound influence on elution time and resolution. In fact, at the lower particle size, resolution and rapidity in data acquisition are of similar quality to those obtained in MECC.13 Moreover, as in the case of the latter technique, all derivatized carbohydrates in the HPLC procedure we developed elute well before the AMAC signal, at about 31 and 27 min using the µBondapak or C18 Waters column, respectively (Figure 1). As in the case of MECC, elution times using reverse-phase HPLC are related to size, so that the largest carbohydrate elutes first and AMAC-glucose, the smallest, elutes last. We had previously determined12 the order of elution in MECC by comparing migration times of the smaller components of the dextran ladder with those of AMAC-derivatized standard carbohydrates. At this stage of this study, the identity of the components of the dextran ladder in the HPLC analysis was obtained by comparing relative peak heights with those obtained in the MECC analysis of the same mixture. These structures were later confirmed by mass spectrometric analysis (see below). The decrease in the overall hydrophobicity of these analytes with an increase in carbohydrate content must play a role in
Figure 2. MECC analysis of (a-d) individual components of the dextran 'adder collected by HPLC (Figure 1) and (e) a mixture of the hydrolyzed dextran ladder before chromatographic fractionation. As in the case of HPLC analysis, the AMAC carbohydrate derivatives elute before AMAC. The signal due to this excess reagent (not shown) is about 15 min after the AMAC-glucose peak (1). Again, numbers refer to the number of glucose units of the solutes analyzed.
determining their order of elution in the HPLC analysis. These results support those published in a recent report,24 where AMACderivatized disaccharides related to chondroitin sulfate were analyzed using an amino column in ion-exchange mode. Under these conditions, excess reagent eluted first and close to the substrates analyzed. The use of sodium hydrogen phosphate in the mobile phase at concentrations up to about 0.8 M does not make this method desirable if chromatographic separation is followed by mass spectrometric analysis. The HPLC method we developed allows collection of AMAC-derivatized oligosaccharides for further studies, without contamination from excess AMAC. To demonstrate this, we collected manually a number of the AMAC polyglucose substrates and reanalyzed by MECC. Results are shown in Figure 2a-d for molecules containing 12, 9, 6, and 4 glucose units linked R-1,6 to one another, respectively. The purity of the individual oligosaccharides is excellent, and, as expected, the order of migration is in good agreement with that observed for the complete dextran pool (Figure 2e). The methodology we developed using AMAC avoids any contamination of derivatized oligosaccharides that range widely in size.18 In fact, the smallest derivative, AMAC-glucose, migrates at least 6 min before excess AMAC. A major advantage of this coordinated HPLC/MECC approach is the identical migration (24) Kitagawa, H.; Kinoshita, A.; Sugahara, K. Anal. Biochem. 1995, 232, 114121.
order of substrates analyzed by the two separation methods; this allows the more facile transfer of information between the two techniques. Moreover, the collection of these pure derivatized oligosaccharides proved to be very useful for structure elucidation, as will be shown later. A major advantage of the present approach, compared to ion-exchange methods, is that no sample cleanup is required. After successfully analyzing the components of the above dextran ladder, we set out to prove the general utility of this approach by analyzing the mannose-rich glycan mixture (Figure 3) from ribonuclease B. Chromatograms obtained using the µBondapak and Symmetry columns are shown in Figure 4. As before, the lower particle size stationary phase gave better resolution and a faster separation time. Again, as in the case of the components of the dextran ladder, preliminary identification of the mannose-rich structures in the HPLC trace was carried out by comparison with the relative intensity of the various components seen in the MECC analysis and previously identified by comigration studies using commercially available standards.13 The three peaks from the µBondapak column were collected manually and then analyzed by MECC. Collection of derivatized glycans from the lower quality separation was carried out to test the robustness of the methodology developed. In cases where the ratio of components could not be achieved due to lack of resolution by HPLC, coordination with mass spectrometry and MECC still allowed determination of these values over a relatively short period of time. The purity of the samples collected is shown in Figure 5b-d, while the resolution of the intact mixture of AMAC-derivatized glycans is shown in Figure 5a. The Man5GlcNAc2 and Man6GlcNAc2 were obtained virtually pure (>90%, Figure 5c,d, respectively), and the Man8GlcNAc2 was considerably enriched (Figure 5b). Considerable improvement in purity should be possible either by collecting more than one fraction from a single peak or by using smaller particle size stationary phases. The purity and identity of the collected samples were confirmed by MALDI mass spectrometry, discussed in a later section. Using the mannose-rich glycan mixture from ribonuclease B, we compared the apparent limits of detection in HPLC (tungsten source) and CE (helium-cadmium laser source). For these experiments, 0.28 µg (∼36 pmol) of the mixture was derivatized. A limit of detection of 5 nmol was obtained with a 3 s dynamic injection in the case of MECC. Increasing the injection time caused too much baseline perturbation. The limit of detection with the tungsten source was about an order of magnitude lower. It is interesting that the order of elution of the mannose-rich glycans in HPLC and that of migration in MECC are again the same, indicating that, as in the case of the derivatized dextran ladder, hydrophobicity plays a major role in the order of elution of these solutes. From a comparison of the retention behavior of the R-1,6-linked glucose units in Figure 1 to that of the glycan pool from ribonuclease B (Figure 4), it appears that the mannoserich structures are more hydrophilic than the polyglucose molecules. As mannose and glucose are isomeric (containing the same number of hydroxy groups), the increased hydrophilicity of the former molecules must be due to the two N-acetylglucose residues in the mannose core structure. Thus, the elution times of Glc3 and Glc8 are equivalent to those of Man5GlcNAc2 and Man8GlcNAc2, respectively. MALDI and Nanospray Mass Spectrometry of HPLCCollected AMAC-Labeled Oligosaccharides. The apparent Analytical Chemistry, Vol. 68, No. 24, December 15, 1996
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Figure 3. Chemical structures of mannose-rich glycans from ribonuclease B.
Figure 4. Chromatographic analysis of glycans derived from ribonuclease B, using (a) µBondapak and (b) Symmetry columns (200 ng of mixture injected). The identity of the mannose-rich structures is as indicated.
correlation13 of hydrodynamic volume to the retention behavior of glycans separated by the HPLC and MECC methodologies discussed above allows evaluation of the relative proportions of the individual glycans in a mixture and some preliminary rationalization about chemical structure. However, considerably more information on chemical identity can be obtained if the separation methodology employed is compatible with spectroscopic and/or spectrometric techniques. HPLC is more amenable to fraction collection and, under the right conditions, allows the mass spectrometric analysis from fractions collected solely from one injection. In this section, we provide examples where we show that chromatographically enriched AMAC-derivatized glycans can be analyzed without difficulty by MALDI or nanospray mass spectrometry. 4428 Analytical Chemistry, Vol. 68, No. 24, December 15, 1996
Figure 5. MECC analysis of (a) glycan pool from ribonuclease B and (b-d) the three individual fractions after chromatographic collection using a µBondapak C18 column (10 ng of mannose-rich mixture injected in the case of (a).
Individual AMAC-derivatized dextran ladder components, purified by HPLC (Figure 2) from a single injection of a mixture (made up of about 22 components by CE and a total mass of about 100 ng), were analyzed by positive ion MALDI-MS. The linear MALDI mass spectrum obtained for the AMAC derivative of maltohexose, shown in Figure 6, is typical. Analysis produces (M + Na)+ and
Figure 6. MALDI mass spectrum of AMAC-maltohexose acquired in linear mode. Table 1. MALDI-MS Data for HPLC Fractions 1-3 Collected from the AMAC-Labeled Mannose-Rich Glycan Pool from Ribonuclease B fraction no.
detected (M + Na)+ (monoisotopic)a
suggested glycan structure
calculated (M + Na)+ (monoisotopic)
1
1937.8 (m) 1775.8 (w) 2099.8 (w) 1613.6 (m) 1775.6 (w) 1451.9
Man8GlcNAc2 Man7GlcNAc2 Man9GlcNAc2 Man6GlcNAc2 Man7GlcNAc2 Man5GlcNAc2
1937.7 1775.6 2099.7 1613.6 1775.6 1451.6
2 3
a Molecular weights shown include AMAC + 2H. m and w signify “main” and “weak” components, respectively.
(M + K)+ ions at m/z 1208 and 1224, respectively, and a much weaker (M + H)+ at m/z 1187.0. These values are within one mass unit of the expected molecular weights. Although minor signals at m/z 1236 and 1253 appear to be related to the sample, since they form adducts of 28 from the corresponding [M + Na]+ and [M + K]+ ions, we do not know their identity at this stage of our studies. The three fractions collected from the HPLC analysis of one injection of the AMAC-labeled glycans from ribonuclease B (Figure 4) were submitted for both MALDI (using the reflectron mode for higher resolution) and nanospray21,22 mass spectrometric analyses. The reflectron MALDI-MS data for the three mannoserich fractions are collected in Table 1. In terms of predicted ribonuclease B glycans, fraction 3 (Man5GlcNAc2) is the purest sample, whereas fraction 1 (Man8GlcNAc2) and fraction 2 (Man6GlcNAc2) are contaminated with small amounts of analogous compounds as well as other components. Figure 7 shows a comparison of typical mass spectra obtained for Man6GlcNAc2 by the two methods. The peaks at m/z 1613.6 (major) and 1775.6 (minor) correspond to the first peak (that is, monoisotopic (M + Na)+) in the resolved molecular ion cluster and represent the (M + Na)+ for Man6GlcNAc2 and Man7GlcNAc2, respectively. These results are in good agreement with the MECC analysis of the collected fractions (Figure 5) in that the levels of enrichment of the collected samples are of the same order by the two methods. Additional peaks are observed in the spectra of this fraction, indicating both lower and higher molecular weight compounds. The higher molecular weight series begins at m/z 1817.8 (monoisotopic), which is 204 Da heavier than the Man6GlcNAc2
Figure 7. Mass spectra acquired from HPLC fraction 2 containing mainly Man6GlcNAc2. (a) MALDI in reflectron mode and (b) nanospray.
component, and continues upward in mass by increments of 162 Da (in-chain mass for Hex). An additional HexNAc would add 203 Da, not 204 Da. Consequently, although these components are apparently carbohydrate in nature, they do not correspond to HexNAc analogs of Man6GlcNAc2, and no further attempts were made to identify them. The peak at m/z 1451.8 in the nanospray mass spectrum corresponds to the loss of Hex from the Man6GlcNAc2. This species is probably formed by fragmentation in the electrospray MS process, as no corresponding component is observed in the MALDI mass spectrum of the same fraction. No attempts were made to identify any of the other minor components of lower molecular weight. In the nanospray spectrum (data not shown) of the Man8 fraction, a major peak observed at m/z 1937.8 corresponds to the (M + Na)+ of Man8GlcNAc2. Weak peaks at m/z 1613.7, 1775.8, and 2099.8 correspond to Man6GlcNAc2, Man7GlcNAc2, and Man9GlcNAc2, respectively. A significant peak is also observed in this spectrum at m/z 1411.7, indicating a nominal molecular weight (assuming the peak corresponds to (M + Na)+) of 1388. Although the identity of this component was not established, the MS/MS spectrum clearly shows that it is carbohydrate-related and that a GlcNAc-AcrH2 group is present. Overall, the molecular weights of the major components determined by the two mass spectrometric methods are in good agreement with one another and with predicted values (Table 1). The data we have presented illustrate the advantages of using AMAC as a label for the rapid analysis of glycan mixtures by both Analytical Chemistry, Vol. 68, No. 24, December 15, 1996
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reverse-phase HPLC and MECC. Using the methodology described, labeled molecules are analyzed over a short period of time without any interference from excess fluorophore, thus avoiding sample-cleaning procedures. In both analytical procedures, unreacted AMAC is retarded or retained by the stationary or the micellar phase due to its higher hydrophobic nature, so that its retention time is well after that of the analytes of interest. We have also shown that one injection of a mixture of glycans in the HPLC system yields enriched fractions that can be analyzed by either nanospray or MALDI mass spectrometry with equal efficiency, and with similar sensitivity (low picomole), thus providing rapid and detailed structural characterization of oligosaccharides derived from glycoproteins. A distinguishing feature of electrospray is the ease with which it can be coupled on-line to either HPLC or CE. We have not presented any online HPLC-MS or CE-MS studies here, and such coupling would probably require modification of the separation protocols used. In the case of analysis by HPLC-MS, the concentration of ammonium acetate in the mobile phase would have to be decreased to below 50 mM in order to maintain reasonable MS sensitivity. In the case of CE-MS, the presence of the deoxycholate surfactant in the buffer is expected to have a deleterious effect on sensitivity of mass spectrometric detection. This may be avoided by fraction collection, and we hope to carry out some studies in this direction.
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The studies we have reported are essentially a proof of concept on the ease and variety of analysis of AMAC-derivatized oligosaccharides. In addition to neutral oligosaccharides, to date we have applied this methodology to a wide range of more complex oligosaccharides, including negatively charged molecules. In fact, HPLC analysis after derivatization with AMAC has been found to be especially useful in the separation and identification of glycans containing sialic acid residues; the order of elution of these molecules is related to the number of these negatively charged sugars attached to the antennal end of the oligosaccharide. These results will be published elsewhere. We are presently conducting extensive studies to derive a strategy for the rapid fingerprinting and structure identification of oligosaccharides using the coordinated approach we have described. In future work, we shall also concentrate on LC electrospray ionization experiments in an attempt to obtain structural information directly, avoiding fraction collection. Preliminary work we have so far carried out indicates that, although this technology is more practicable and rapid, the sensitivity of detection is lower than that of the indirect methods we have described. Received for review July 22, 1996. Accepted October 3, 1996.X AC960721+ X
Abstract published in Advance ACS Abstracts, November 1, 1996.
