Anal. Chem. 1998, 70, 3840-3844
Capillary Liquid Chromatographic and Automated MALDI-TOF Mass Spectrometric Analysis of Complex Carbohydrate Mixtures Rudi Grimm,† Helen Birrell,‡ Gordon Ross,† Joanne Charlwood,‡ and Patrick Camilleri*,‡
Hewlett-Packard GmbH, Chemical Analysis GroupsEurope, Strasse 8, 76337 Waldbronn, Germany, and SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, U.K.
Reproducible microbore reversed-phase HPLC methodology has been developed, suitable for the analysis of carbohydrates derivatized with 2-aminoacridone. The high extinction coefficient of this label at around 260 nm, combined with the use of a new diode array microflow cell, has allowed detection at femtomole levels. Analytes were also robotically collected onto MALDI targets, and mass spectrometric information was acquired for carbohydrate present at low picomole levels. This technology was successfully applied to mixtures of linear and branched oligosaccharides, including sialylated species. Unlike the biosynthesis of nucleic acids and proteins, carbohydrate processing requires a different enzyme at each step, and each product becomes an exclusive substrate for the next enzyme in the processing pathway. The extensive heterogeneity of glycans is mainly due to a number of glycosidases and glycosyltransferases that sequentially act on the glycoprotein either as the polypeptide chain enters the endoplasmic reticulum or in the cisternal compartments of the Golgi apparatus. Despite this complex processing pathway, it is often observed that the molar ratio of glycans released from glycoproteins from healthy individuals is constant.1 Variations in carbohydrate mixtures often occur in patients due to variations in glycosidase and/or glycosyltransferase expression. These changes can be useful in the noninvasive and early diagnosis of disease.1,2 Relating glycan structure to biological function can also be important in the design of potential therapeutic agents.3 Unfortunately, unlike the case of nucleic acids, where a polymerase chain reaction (PCR) replication system is frequently used to amplify minute amounts of material, no equivalent system exists in the case of carbohydrate. Thus, structural analysis of these compounds has, until recently, only been possible by the often laborious collection of enough compound for radiolabeling, followed by low-resolution size-exclusion chromatography.4 The lack of a suitable natural chromophore or fluorophore in the structure of the majority of glycans of biological interest has meant †
Hewlett-Packard GmbH. SmithKline Beecham Pharmaceuticals. (1) Varki, A. Glycobiology 1993, 3, 97-130. (2) Rahbek-Nielsen, H.; Roepstorff, P.; Reischl, H.; Wozny, M.; Koll, H.; Haselbeck, A. J. Mass Spectrom. 1997, 32, 948-958. (3) Cumming, D. A. Glycobiology 1991, 1, 115-130. (4) Dwek, R. A. Chem. Rev. 1996, 96, 683-720. ‡
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that direct absorbance or fluorescence detection has not been possible. More recent developments in high-resolution high-performance liquid chromatographic (HPLC) techniques and the application of intense fluorophores as highly efficient derivatizing agents has permitted the analysis of nano- and picomole levels of oligosaccharide mixtures.5,6 We have reported the use of 2-aminoacridone (2-AMAC) for the successful resolution of complex carbohydrate mixtures by micellar electrokinetic capillary chromatography (MECC)7-9 and HPLC.10,11 This derivatizing agent also makes carbohydrates amenable to MALDI-TOF and electrospray mass spectrometric analysis. In the present study, we report the separation of a number of 2-AMAC-derivatized linear and branched oligosaccharides by capillary reversed-phase HPLC. The high extinction coefficient of 2-AMAC at a wavelength around 260 nm and the use of a newly developed microflow cell has allowed the analysis of individual analytes present at a level below 100 fmol. Automated collection of HPLC fractions onto a MALDI-TOF target reduced handling of samples and provided molecular weight information for neutral and sialylated oligosaccharides. EXPERIMENTAL SECTION Materials. Sodium cyanoborohydride, ammonium acetate, dimethyl sulfoxide, and trifluoroacteic acid were supplied by Aldrich (Poole, U.K.). Glacial acetic acid was purchased as ANALAR grade from Fisher Scientific (Loughborough, U.K.). 2-AMAC was purchased from Fluka (Gillingham, U.K.) and was used without further purification. Aqueous solutions were prepared using distilled-deionized water. The enzyme-hydrolyzed dextran ladder and glycan mixtures from bovine ribonuclease B and human IgG were obtained from Oxford GlycoSystems (Abingdon, U.K.). (5) Anumula, K.; Taylor, B. P. Eur. J. Biochem. 1991, 195, 269-280. (6) Guile, G. R.; Rudd, P. M.; Wing, D. R.; Prime, S. B.; Dwek, R. A. Anal. Biochem. 1996, 240, 210-226. (7) Greenaway, M.; Okafo, G. N.; Camilleri, P.; Dhanak, D J. Chem. Soc., Chem. Commun. 1994, 1691-1692. (8) Camilleri, P.; Harland, G. B.; Okafo, G. Anal. Biochem. 1995, 230, 115122. (9) Okafo, G. N.; Burrow, L. M.; Neville, W.; Truneh, A.; Smith, R. A. G.; Camilleri, P. Anal. Biochem. 1996, 240, 68-74. (10) Okafo, G.; Burrow, L.; Carr, S. A.; Roberts, G. D.; Johnson, W.; Camilleri, P. Anal. Chem. 1996, 68, 4424-4430. (11) Okafo, G.; Langridge, J.; North, S.; Organ, A.; West, A.; Morris, M.; Camilleri, P. Anal. Chem. 1997, 69, 4985-4993. S0003-2700(98)00235-2 CCC: $15.00
© 1998 American Chemical Society Published on Web 08/20/1998
Figure 2. Analysis of a 0.6-ng mixture of a dextran ladder.
Figure 1. (a) Separation of the components of a dextran ladder and spectral analysis of 2-AMAC and peak 13. (b) Expansion of the time scale between 18 and 30 min and spectral analysis of low-level peaks 20 and 22.
Derivatization of Carbohydrate Mixtures with 2-AMAC. Reductive amination of the carbohydrate mixtures was carried out in an aqueous DMSO/glacial acetic acid mixture and in the presence of sodium cyanoborohydride, as detailed by us previously.7-11 Capillary HPLC. An HP 1100 system (Hewlett-Packard, Waldbronn, Germany) was used as described recently.12 In the present study, an HP G1315 diode array detector was fitted with a new diode array microflow cell (10-mm path length, 500-nL volume). Separations were performed at room temperature on a 250- × 0.3-mm modified C-18 column (LC Packings, The Netherlands) with a flow rate of 2 or 4 µL/min, using a gradient from 10 to 25% B within 35 min and remaining at 25% B for a further 20 min for the lower flow rate, and from 10 to 25% B within 45 min for the higher flow rate. Solvent A was 100 mM ammonium acetate (pH 6.5) and solvent B 100% acetonitrile. Signals were recorded at a wavelength of 260 nm. In addition, spectra were collected from 200 to 500 nm. Fraction Collection and MALDI-TOF MS Analysis. Automated on-line microfraction collection of 1-µL fractions was done robotically onto MALDI targets using the BAI Probot microfraction collector as previously described.12 The matrix consisted of 2,5-dihydroxybenzoic acid and 0.1% trifluoroacetic acid. MALDITOF MS analysis was carried out on the HP G2030A system (Hewlett-Packard, Palo Alto, CA). RESULTS AND DISCUSSION Previously, we had shown that 2-AMAC-derivatized oligosaccharides can be separated by reversed-phase HPLC, followed by fluorescence detection.10 This methodology involved the manual collection of samples into appropriate containers by visually (12) Grimm, R.; Serwe, M.; Chervet, J. P. LC-GC 1997, 960-968.
Figure 3. MALDI-TOF MS of automatically collected fractions: (a) components 7 and 8; (b) components 13 and 14.
Figure 4. Microbore capillary LC analysis of a mixture of glycans released from ribonuclease B. The numbers on peaks are related to the mass spectra in Figure 6 and the structures in Table 1.
following the progress of a chromatographic run. After lyophilization, glycan residues were mixed with an appropriate matrix before analysis by MALDI-TOF mass spectrometry. As the molecular weight information on the analytes analyzed by this technique was of excellent quality (both in signal-to-noise ratio and molecular weight accuracy), we carried out further studies aimed at reducing the amount of sample analyzed and attempted to avoid, as much as possible, sample handling after chromatography. To achieve Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
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Figure 6. Microbore capillary LC analysis of a mixture of glycans released from IgG. The numbers on peaks are related to the mass spectra in Figure 7 and the structures in Table 2.
Figure 5. Mass spectral analysis of fractions collected from the microbore capillary LC analysis of a mixture of glycans released from ribonuclease B (see Figure 5).
these objectives, we chromatographed glycan mixtures using a capillary C-18 column. Fractions were then automatically collected by a robotic system and analyzed by MALDI-TOF mass spectrometry. Figure 1a shows the analysis of a 120-ng mixture of commercially available enzymatically hydrolyzed dextran. For these measurements, the flow rate of the mobile phase was 2-µL/min, and the 2-AMAC-derivatized carbohydrates were monitored by following their ultraviolet absorbance at a wavelength of 260 nm. The extinction coefficient of 2-AMAC is about 40 000 at this wavelength. A further increase in the sensitivity of detection was achieved by the use of a microflow cell which contained a highly reflecting interior. 3842 Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
The peak around 63 min in Figure 1a is due to excess 2-AMAC used in the derivatization reaction. The number on the peaks refers to the number of glucose units covalently linked to 2-AMAC. As the mechanism of elution is largely dependent on the relative hydrophobicity of the analytes, the glucose derivative has the longest retention time, whereas increasing the number of glucose units decreases hydrophobicity and, hence, the resulting retention time. Enlargement of the chromatogram from 18 to 30 min (Figure 1b) shows that the total number of 2-AMAC-derivatized glycans is at least 22. The UV-visible spectrum of all signals over the wide wavelength region of 200-500 nm was obtained using a diode array detector. Spectra for 2-AMAC and analytes containing 12, 20, and 22 glucose units are shown as insets in Figure 1. From these data, it is clear that covalent attachment of sugar units to 2-AMAC does not cause a shift in the wavelength for the absorbance maxima. This property is very useful, as it allows preliminary information to be obtained about the relative proportion of the various components in a mixture. Figure 2 shows the analysis of a total amount of 0.6 ng of the same mixture of the dextran ladder. A 4 µL/min flow rate was used for these measurements. About 11 components could be seen under these conditions. From the ratios of peak heights, we estimate that the amount of the fastest component was well below 60 fmol. Fractions of eluent from the analysis of the 120-ng injection (Figure 1) were robotically collected onto MALDI targets, containing a matrix consisting of 2,5-dihydroxybenzoic acid and 0.1% trifluoroacetic acid. Typical mass spectra are shown in Figure 3. As expected, adjacent analytes differed by 162 mass units. For the 2-AMAC derivatives present at the higher concentrations, such as the two shown in Figure 3a, sodium and potassium adducts, [M + Na]+ and [M + K]+, were observed in addition to the protonated species, [M + H]+. In the case of the lower concentration components, only alkali metal ion adducts could be distinguished from baseline noise. The successful analysis of linear oligosaccharides already described encouraged us to apply this microbore separation technique to the analysis of branched carbohydrates. We chose the mixtures of glycans released from two glycoproteins, ribonu-
Figure 7. Mass spectral analysis of fractions collected from the microbore capillary LC analysis of a mixture of glycans released from IgG (see Figure 6).
clease B and IgG. The first mixture is known to contain only high-mannose structures,10 whereas the second mixture contains sialylated and nonsialylated complex glycans.13 A total of 300 ng of each mixture was injected on to the microbore column. Chromatography of the high-mannose glycans gave three wellresolved peaks (1, 3, and 4) and a shoulder (2) (Figure 4). Similar to the case of the 2-AMAC-derivatized dextran ladder components, the order of elution was expected to be related to the number of mannose units at the nonreducing end of these glycans. This was confirmed by MALDI-TOF mass spectrometric analysis of collected fractions, as shown in Figure 5; fractions 4 and 3 (13) Rothman, R. J.; Warren, L.; Vliegenthart, J. F. G.; Hard, K. J. Biochemistry 1989, 28, 1377-1384.
consisted almost exclusively of Man5 and Man6, respectively; fraction 2 contained Man7 as the main component and Man8; fraction 1 was considerably enriched in Man8 and contained Man9 as the minor component. Chemical structures of Man 5 to Man 9 and molecular weights corresponding to their 2-AMAC derivatives are given in Table 1. Analysis of a mixture of glycans released from IgG gave the chromatogram shown in Figure 6. The numbered peaks were again analyzed by MALDI-TOF mass spectrometry, and the corresponding spectra are shown in Figure 7. Peak 1 consisted of a mixture of two biantennary glycans containing two sialic acid residues; the difference in the molecular weight of these components was 202 mass units, indicating that the higher mass glycan Analytical Chemistry, Vol. 70, No. 18, September 15, 1998
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Table 1. Positive Ion Mode MALDI-MS Analysis of Derivatized Glycans Released from Ribonuclease Ba
a Key to structure: mannose, b; N-acetylglucosamine, 9. Molecular weights shown include 2-AMAC + 2H. Molecular ions refer to (M + Na)+. The asterisk indicates carryover from peak 1.
Table 2. Positive Ion Mode MALDI-MS Analysis of Derivatized Glycans Released from Human IgGa
a Key to structure: mannose, b; galactose, 4; N-acetylglucosamine, 9; fucose, 0; sialic acid, O. Molecular weights shown include 2-AMAC + 2H. Molecular ions refer to (M + Na)+.
contained a bisecting N-acetylglucosamine residue. The main component in peak 2 differed from one of the sialylated glycans in peak 1 by 291 mass units; a monosialylated N-acetylglucosamine (14) Furukawa, K.; Kobata, A. Mol. Immunol. 1991, 28, 1333-1340. (15) Gianazza, E. J. Chromatogr. A 1995, 705, 67-87.
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analogue with a molecular weight of 2499 was also present in this fraction. The observed order of elution of disialylated molecules and the less hydrophobic monosialylated molecules is as expected under the present chromatographic conditions. Other minor and lower molecular weight components in this fraction are almost certainly due to hydrolysis of the main glycans caused by the acidity of the matrix. The molecular weight (1859.9) of the major component in fraction 3 agrees with the structure of a non-core-fucosylated glycan. This fraction is contaminated with the components in peak 2. Addition of a fucose residue (146 mass units) to this structure gives the molecular weight of 2005 measured for the component in fraction 4. Other 2-AMAC derivatives of core-fucosylated neutral glycans were identified in fractions 5 and 6. The molecular weights in fractions 4-6 differed by 162 mass units, which corresponds to the molecular weight of a galactose residue. These deductions on the structures of the glycans from IgG were made from a knowledge of published data,13,14 as it is not possible to deduce the type and linkage of sugar residues only from molecular weight information. Table 2 shows the structures of the glycan derivatives identified and the molecular weights of the corresponding 2-AMAC derivatives. In conclusion, we have shown that reversed-phase chromatography allows the partial resolution and enrichment of the components of complex linear and branched oligosaccharide mixtures. The method developed avoids handling of samples from collection to MALDI-TOF analysis, so that the elimination of usually labile sugar residues such as sialic acid15 before mass spectrometric analysis is reduced considerably. From peak area measurements, we calculate that the two disialylated residues in the first-eluting component (peak 1 in Figure 6) were present at a level of about 10 pmol. Our future work will concentrate on the development of chromatographic methodology with greater resolving power. We also intend to couple microbore chromatography to electrospray mass spectrometry; MS-MS analysis by this technique will enable the aquisition of more data related to the sequence of glycans under study. Received for review March 4, 1998. Accepted June 29, 1998. AC980235H
