Anal. Chem. 1997, 69, 4985-4993
High-Performance Liquid Chromatographic Analysis of Complex N-Linked Glycans Derivatized with 2-Aminoacridone George Okafo,† James Langridge,‡ Simon North,† Andrew Organ,† Andrew West,† Mike Morris,‡ and Patrick Camilleri*,†
SmithKline Beecham Pharmaceuticals, Coldharbour Road, Harlow, Essex, CM19 5AD, U.K., and Micromass Limited, Floats Road, Wythenshawe, Manchester M23 9LZ, U.K.
2-Aminoacridone (2-AMAC) has been used to derivatize mixtures of N-linked oligosaccharides released from r1acid glycoprotein and immunoglobulin G. In each case, the HPLC profile obtained for the derivatized glycans was compared to that obtained after digestion with sialidase and a two-enzyme array system made up of sialidase and r-fucosidase, prior to derivatization by 2-AMAC. These studies are rapid and provide a wealth of preliminary information about the degree of sialylation and core fucosylation in the corresponding parent glycans. Moreover, collection of glycans from one single injection has provided enough material for molecular weight determination by MALDI-MS analysis. In this study we have also carried out limited MS-MS studies on enriched fractions of 2-AMAC-glycans using a nanospray orthogonal quadrupole time-of-flight mass spectrometer.
Pulsed amperometric detection at high alkaline pH is often used in the analysis of oligosaccharides.6 As these analytes do not usually absorb above a wavelength of ∼200 nm, analysis is often carried out indirectly following derivatization with a suitable chromophore or fluorophore.7-9 Recently we introduced the use of 2-aminoacridone (2-AMAC) as a sensitive fluorophore for the detection of monosaccharides10 and neutral polysaccharides.11-13 We have also shown that a 2-AMAC-derivatized mixture of linear polysaccharides and a mixture of mannose-rich glycans from ribonuclease B were amenable to analysis by both matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and nanospray mass spectrometry.14 2-AMAC-glycan derivatives are produced by a “one-pot” Schiff reaction at the reducing end (that is, the N-acetylglucosamine residue) followed by reduction and can be represented by the structure
It is increasingly being recognized that oligosaccharides can play an important role in the structure and function of glycoproteins1 and can affect protein stability and folding.2 Changes in intercellular recognition by a protein may be affected by the absence or nature of its glycosylation.3 Glycans can also influence the pharmacokinetics and functional activity of a therapeutic protein.4 Glycans linked to the amide group of an asparagine residue (usually termed as N-glycans) are the predominant protein-carbohydrate modifications in eukaryotic cells.5 Although N-glycans can be classified into three main groups, high mannose, complex, and hybrid, their structures can vary enormously. The core portion (reducing end) of N-glycans is common to all and is composed of two N-acetylglucosamine and three mannose residues. Variation in glycan structure occurs in several ways, some of which include (a) the presence of a fucose residue attached to the end N-acetylglucosamine, (b) variation in the number and length of side chains emanating from the core mannose residues, and (c) sialylation of the terminal galactose residues.
In this study, we have used the same HPLC methodology for the analysis of more complex oligosaccharides released from the glycoproteins R1-acid glycoprotein (R-AGP) and immunoglobulin G (IgG). To identify glycans that contain either one or more sialic acid residues or core fucose, we have devised a protocol involving treatment of glycan mixtures with sialidase and with a two-enzyme array made up of sialidase and R-fucosidase, prior to derivatization
†
SmithKline Beecham Pharmaceuticals. Micromass Limited. (1) Varki, A. Glycobiology 1993, 3, 97-130. (2) Dwek, R. A. Chem. Rev. 1996, 96, 683-720. (3) Bollensen, E.; Schachner, M. Neurosci. Lett. 1987, 82, 77-82. (4) Couser, W. G.; Johnson, R. J.; Young, B. A.; Yeh, C. G.; Toth, C. A.; Rudolph, A. R. J. Am. Soc. Nephrol. 1995, 5, 1888-1894. (5) Kornfeld, R.; Kornfeld, S. Annu. Rev. Biochem. 1985, 3, 97-130. ‡
S0003-2700(97)00713-0 CCC: $14.00
© 1997 American Chemical Society
(6) Hayase, T.; Dziegielewska, K. M.; Kuhlenschmidt, M.; Reilly, T.; Lee, Y. C. Biochemistry 1992, 31, 4915-4921. (7) Honda, S.; Makino, A.; Suzuki, S.; Kakehi, K. Anal. Biochem. 1990, 191, 228-234. (8) Chiesa, C.; Horvath, C. J. Chromatogr. 1993, 645, 337-352. (9) Suzuki-Sawada, J.; Umeda,Y.; Kondo, A.; Kato, I. Anal. Biochem. 1992, 207, 203-207. (10) Greenaway, M.; Okafo, G. N.; Camilleri, P.; Dhanak, D. J. Chem. Soc., Chem. Commun. 1994, 1691-1692. (11) Camilleri, P.; Harland, G. B.; Okafo, G. Anal. Biochem. 1995, 230, 115122. (12) Harland, G. B.; Okafo, G.; Matejchuk, P.; Sellick, G. E.; Chapman, G. E.; Camilleri, P. Electrophoresis 1996, 17, 406. (13) Okafo, G. N.; Burrow, L. M.; Neville, W.; Truneh, A.; Smith, R. A. G.; Camilleri, P. Anal. Biochem. 1996, 240, 68-74. (14) Okafo, G. N.; Burrow, L. M.; Carr, S. A.; Roberts, G. D.; Johnson, W.; Camilleri, P. Anal. Chem. 1996, 68, 4424-4430.
Analytical Chemistry, Vol. 69, No. 24, December 15, 1997 4985
Figure 1. HPLC analysis of AMAC-glycans from human R-glycoprotein. The signals shown in (a) are primarily due to AMAC derivatives of acidic glycans. Treatment of glycans from this glycoprotein with sialidase and a combination of sialidase and fucosidase, prior to AMAC derivatization gives (b) and (c), respectively. Numbers shown refer to fractions collected for MALDI and MS-MS measurements.
with 2-AMAC. Although the procedure developed does not provide complete information about the range of structures in a glycan mixture, it is valuable in the rapid analysis of the main components and gives a preliminary estimate of the extent of sialylation and core fucosylation. Fractions collected from one single HPLC injection have provided sufficient material for both MALDI-MS spectrometric and MS-MS electrospray sequence analysis. EXPERIMENTAL SECTION Chemicals. Glacial acetic acid and acetonitrile were obtained from BDH (Poole, U.K.). Dimethyl sulfoxide (DMSO), ammonium acetate, and sodium cyanoborohydride were purchased from Aldrich (Gillingham, U.K.). The derivatization reagent 2-aminoacridone was synthesized according to the method outlined by Barnett et al.15 The crude product was recrystallized from ethyl acetate, and petroleum ether was added slowly to precipitate the product. Final purification was carried out by preparative HPLC using the following conditions: Dynamax C-18 reversedphase column (190 × 30 mm (i.d.)); mobile phase, acetonitrile/ water, 40:60); flow rate, 4 mL/min; fluorescence detection (excitation wavelength 428 nm, emission wavelength, 525 nm). The collected solution was lyophilized using a GeneVac (Ipswich, U.K.) freeze-drying system to give a yellow solid free of residual ammonium acetate. (15) Barnet, M. M.; Gillieson, A. H. C.; Kermack, W. O. J. Chem. Soc. 1934, 433-435.
4986 Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
Glycan mixtures from R-AGP and IgG, oligosaccharide standards, and enzymes were purchased from Oxford GlycoSystems (Abingdon, U.K.). Derivatization with 2-AMAC. Carbohydrates were derivatized with 2-AMAC according to the procedure reported by Jackson.16 The derivatization involved a weight (∼1 µg) of dried lyophilized glycan mixture, to which was added a volume (10 µL) of 0.1 M 2-AMAC solution (2 mg of the solid dissolved in a 3:17 (v/v) mixture of glacial acetic acid and DMSO, respectively) in an 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 200 mL of 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 solid carbon dioxide bath at -20 °C. An aliquot of the 2-AMAC solution was diluted and analyzed by reversed-phase HPLC. Enzymic Digestion of Oligosaccharides. For the digestion with sialidase (Vibrio cholerae), an aliquot (10 µL) of the enzyme solution was added to an Eppendorf tube containing the dried glycan (∼40 µg) and manually agitated for 30 s. The reaction tube was then incubated at 37 °C for 18 h. Another volume (10 µL) of the enzyme solution was then added and the tube incubated for a further 2 h. After lyophilization, the dried reaction mixture was derivatized with 2-AMAC. In the case of the simultaneous digestion with fucosidase (bovine epididmyis) and sialidase, the reaction procedure was similar except that aliquots (10 µL) of both enzyme solutions were added to the same sample tube. Reversed-Phase HPLC Conditions. Chromatographic separation was performed using a Perkin Elmer Series 4 liquid chromatograph which was fitted with a Waters C-18 Symmetry column (4.6 × 250 mm). About 100 ng of the mixture of 2-AMAC derivatives was injected on to the column and detected by fluorescence using the Perkin Elmer LS-4 fluorescence spectrometer (λex ) 442 nm and λem ) 520 nm) with a slit width of 10 nm. The mobile phase comprised 100mM 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). These were concentrated to near-dryness using a GeneVac centrifuge (Ipswich, U.K.) and reconstituted to ∼5 µL with water before submission for MALDI-MS measurements. MALDI-MS. Spectrometric analysis of the derivatized glycans was carried out using a TofSpec SE mass spectrometer (Micromass, Manchester, U.K.) operated in either the reflectron (glycans from R-AGP) or linear (glycans from IgG) modes. Photon irradiation from a 337-nm pulsed nitrogen laser and 25-keV accelerating potential was used. The instrument was externally mass calibrated using the molecular ions (M + H)+ from a mixture of the two peptides ACTH 18-39 and substance P. A matrix solution was prepared by dissolving 7.5 mg of 2,5-dihydroxybenzoic 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 2-AMAC-glycans collected from HPLC were reconstituted in 15 µL of 20% acetonitrile in water. A 0.5-µL aliquot of sample and of matrix was spotted on a stainless steel target, and the solvent was (16) Jackson, P. Anal Biochem. 1991, 196, 238-244.
quickly evaporated with a stream of argon. Derivatized glycans were detected as the (M + Na)+ and the (M + K)+ ions. MS-MS Analysis. Electrospray MS and MS-MS data were acquired on a QTOF instrument (Micromass, Manchester, U.K.). This hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer was fitted with a nanoflow electrospray ion source. Samples of derivatized glycans obtained from HPLC fractionation were redissolved in 5 µL of a solution containing an equal amount of methanol and 0.2% formic acid. A 2-µL sample of this solution was loaded into borosilicate nanoflow tips. The mass spectrometer was operated with a source temperature of 30 °C and a drying gas flow of 40 L/h. A potential of 1.5 kV applied to the nanoflow tip in the ion source, combined with a nitrogen back pressure of 5-6 psi, produced a flow rate of about 10-20 nL/min into the analyzer. Data were acquired in continuum mode with an integration time of 10 s. For the acquisition of MS data, the quadrupole was used in the radio frequency mode and transmitted about a decade in mass to the TOF. For MSMS studies, the quadrupole was used to select the parent ion, which was subsequently fragmented in the collision cell using argon at a pressure of ∼3 × 10-5 mbar and an appropriate collision energy. RESULTS AND DISCUSSION Separation of 2-AMAC Derivatives by RP-HPLC. Panels a-c of Figure 1 show chromatograms of the 2-AMAC derivatives of the intact glycan mixture from R-AGP and after digestion with sialidase and a mixture of sialidase and R-fucosidase, respectively. This glycan mixture was found to be very heterogeneous in agreement with other authors.17-19 Moreover, treatment with sialidase (Figure 1b) led to a simplification of the chromatographic profile, indicating that the major oligosaccharides in R-AGP contain one or more sialic acid residues. Using the reversed-phase HPLC methodology outlined in the Experimental Section, it was possible to obtain an idea about the type of glycans in this complex mixture from retention time characteristics. 2-AMAC is considerably more hydrophobic than its glycan derivatives so that the retention time of excess fluorophore is longer than that of the carbohydrate derivatives. In the case of the 2-AMAC derivatives of neutral glycans, the order of elution is roughly related to the number of sugar residues; thus, the larger glycans elute before the smaller components of a derivatized glycan mixture. Using the same rationale, it is expected that sialylated glycans have a shorter retention time than the corresponding neutral parent molecules, and the greater the extent of sialylation the shorter is retention on a reversed-phase column. In agreement with these predictions, panels a and b of Figure 1 show that the sialylated glycans of R-AGP have a shorter retention time than the corresponding desialylated molecules. We made no attempt to identify the individual components in the intact R-AGP glycan mixture. However, from the retention times observed, it was apparent that multiantennary sialylated oligosaccharides were present. This agrees with a study19 of the glycan structures of R-AGP purified from liver metastases of (17) Fournet, B.; Montreuil, J.; Strecker, G.; Dorland, L.; Haverkamp, J.; Vliegenthart, J. F. G.; Binette, J. P.; Schmid, K. Biochemistry 1978, 17, 52065214. (18) Yoshima, H.; Matsumoto, A.; Mizuochi, T.; Kawasaki, T.; Kobata, A. J. Biol. Chem. 1981, 256, 8476-8484. (19) Chandrasekaran, E. V.; Davila, M.; Nixon, D.; Medicino, J. J. Cancer Res. 1984, 44, 1557-1567.
primary colon, lung, and breast tumors, which revealed that a large part of the glycans had various sialylated forms of the triantennary and tetraantennary structures I and II, respectively. A smaller proportion were sialylated forms of the bi-antennary structure III. Taverna et al.20 reported that native R-AGP contained mono-, di-, tri-, and tetrasialylated glycans.
Peak 4 in the mixture of the 2-AMAC derivatives of the desialylated glycans was confirmed as III by co-injection with a derivatized standard. The shorter retention time of the other components was a good indication that the corresponding structures were more complex and probably related to I and II. No appropriate triantennary and tetraantennary glycan standards were available, so that peaks 1-4 in Figure 1b were collected from an analytical run and structural inference was done by molecular weight measurement using MALDI mass spectrometry (see later). Simultaneous digestion of the R-AGP glycan mixture with sialidase and R-fucosidase did not lead to any changes in the retention times of the four major peaks (Figure 1c), suggesting that the level of core fucosylation in this glycoprotein was low. However, the small changes in peak intensities, together with the appearance of a relatively low signal at ∼23 min (related to the 2-AMAC derivative of fucose) was an indication of the presence of a low level of core-fucosylated glycans in R-AGP. These results were again in agreement with literature findings.17,18 Fournet et al.17 have reported that R-AGP from normal human serum contained di-, tri-, and tetraantennary complex-type oligosaccharide chains which were not core-fucosylated. Another investigation by Yoshima et al.18 showed again that core-defucosylated tetraantennary glycan structures were present in excess of 90% in plasma (20) Taverna, M.; Baillet, A.; Biou, D.; Schulter, M.; Werner, R.; Ferrier, D. Electrochemistry 1992, 13, 359-366.
Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
4987
Figure 3. MALDI analysis of (a) fraction 2 and (b) fraction 3 for glycans from human R-AGP (see Figure 1b).
Figure 2. HPLC analysis of AMAC-derivatized glycans from human IgG: (a) untreated with enzyme, (b) treated with sialidase only, and (c) treated with a mixture of sialidase and fucosidase. Numbers shown refer to fractions collected for mass spectrometry measurements.
R-AGP. Both groups desialylated the glycan mixture before analysis and did not provide the extent of sialylation in R-AGP. We also applied the above HPLC protocol to the analysis of a glycan mixture released from human serum IgG. The main glycan structures expected from this glycoprotein are biantennary containing a fucose residue linked to N-acetylglucose at the reducing end.21,22 In addition, oligosaccharides containing a bisecting N-acetylglucoseamine residue usually account for 1020% of the total. Core-fucosylated glycans are also present at a higher concentration than the nonfucosylated analogues. Rapid evaluation of the distribution of these classes of glycans is thought to be important for the early diagnosis of rheumatoid arthritis (RA).23 The HPLC analysis of the 2-AMAC derivatives from an IgG glycan pool is shown in Figure 2a. The three major components of the glycan mixture from IgG have been reported21,22 as the corefucosylated structures IV-VI and the monosilylated forms of IV. Using standards peaks, 4-6 were identified as IV-VI. The lowest intensity peak 1 was related to the di-sialylated form of IV. We did not have standards for the two monosialylated forms of IV. (21) Furukawa, K.; Kobata, A. Mol. Immunol. 1991, 28, 1333-1340. (22) Takahashi, N.; Ishii, I.; Ishihara, H.; Mori, M.; Tejima, S.; Jeffries, R.; Endo, S.; Arata, Y. Biochemistry 1987, 26, 1137-1144. (23) Bodman-Smith; K., Sumar, N.; Sinclair, H.; Roitt, I.; Isenberg, G.; Young, A. Br. J. Rheumatol. 1996, 35, 1063-1066.
4988
Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
Peaks 2 and 3 were assigned to these glycans as these were expected to elute at a retention time between the neutral and the disialylated glycans. The fact that these peaks were related to sialylated species was also confirmed by enzyme digestion studies, as will be shown in the next paragraph. Although the human source of the IgG used in this study was not known and variations might be expected from different sources, the elution intensity profile of the main components obtained with 2-AMAC bore similarity to that reported recently using 2-aminobenzamide derivatization.24 As in the case of R-AGP, the underivatized glycan pool released from IgG was subjected to digestion with sialidase and a combina(24) Guile, G. R.; Rudd, P. M.; Wing, D. R.; Prime, S. B.; Dwek, R. A. Anal Biochem. 1996, 240, 210-226.
Figure 4. Maximum entropy-corrected MALDI spectra for fraction 1 from human R-AGP. derivatization with 2-AMAC was carried out after glycans were treated with (a) sialidase and (b) a mixture of sialidase and R-fucosidase. Table 1. MALDI-MS Data for HPLC Fractions 1′-4′ (Figure 1c) Collected from the 2-AMAC-Labeled Glycan Pool from r-AGP, Digested with Sialidase and r-Fucosidase Prior to Derivatization fraction no.
detecteda (M + Na)+ (monoisotopic)
suggested glycan structure
calculated (M + Na)+ (monoisotopic)
1
2735 (m1)b 2954 (m2) 2881 (l) 3100 (l)c 3319 (l)c 2589 (m1) 2370 (m2) 2223 1858
Man3GlcNAc6Gal4 Fuc Man3GlcNAc7Gal5 Man3GlcNAc6Gal4 Fuc2 Man3GlcNAc7Gal5 Fuc Man3GlcNAc8Gal6 Man3GlcNAc6Gal4 Man3GlcNAc5Gal3 Fuc Man3GlcNAc5Gal3 Man3GlcNAc4Gal2
2735 2954 2881 3100 3319 2588 2369 2223 1858
2 3 4
a Molecular weights shown include AMAC + 2H. b m , m , and l signify major, minor, and low-level components, respectively. c These molecules 1 2 are probably core-fucosylated in the original glycan mixture.
Table 2. MALDI-MS Data for (a) HPLC Fractions 4-6 Collected from the AMAC-Labeled Glycan Pool from Human IgG Digested with Sialidase and (b) Fractions 1′, 2′, and 3′ after Digestion with Sialidase and r-Fucosidase Prior to Derivatization
a
fraction no.
detecteda (M + Na)+ (monoisotopic)
suggested glycan structure
calculated (M + Na)+ (monoisotopic)
4 5 6 1′ 2′ 3′
2005 1843 1680 1858 1697 1534
Man3GlcNAc4Gal2 Fuc Man3GlcNAc4Gal1 Fuc Man3GlcNAc4 Fuc Man3GlcNAc4Gal2 Man3GlcNAc4Gal1 Man3GlcNAc4
2004 1842 1680 1858 1696 1534
Molecular weights shown include AMAC + 2H.
tion of sialidase and fucosidase. This was again followed by derivatization with 2-AMAC and HPLC analysis without enzyme removal. Results of this analysis are shown in Figure 2b and c. Sialidase treatment resulted in a decrease in the peaks 1-3 and a resulting increase in the earliest eluting major signal related to (IV) (Figure 2b). From these data, a rough estimate for the degree of sialylation was found to be about 10-15%, in agreement with literature findings.21 Desialylation also led to the appearance
of minor peaks below 15 min. Peak-matching experiments using appropriate standards and defucosylation of the glycan mixture prior to derivatization with IgG showed that these signals coincided with the positions of elution of the defucosylated forms of glycans IV-VI. Unlike desialylation, defucosylation led to considerable changes in the chromatogram (compare Figure 2b and c). The appearance of a high-intensity peak at ∼24 min in Figure 2c confirmed that Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
4989
Figure 5. MALDI spectra of AMAC glycans related to human IgG. The spectrum in (a) is for fraction 5 in Figure 2b and the spectrum in (b) is for fraction 2′ in Figure 2c.
the large majority of oligosaccharides in the glycan mixture from IgG are core-fucosylated. Further comparison of Figure 2b and c also showed that the three major peaks in the defucosylated sample had moved to a shorter retention time. As it was alluded before, these glycans were identified as the defucosylated forms of IV-VI. Final confirmation of the identity of these glycans was obtained by MALDI-MS measurements, as will be shown in the final section. It is known that small amounts (
