Glycoprotein Characterization Combining Intact Protein and Glycan

In this sense, the primary structure of N-glycans is defined by number of antennas, sialylation degree, presence of fucoses, ...... Cloud Computing in...
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Anal. Chem. 2006, 78, 5384-5393

Glycoprotein Characterization Combining Intact Protein and Glycan Analysis by Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry Elvira Balaguer† and Christian Neusu 1 ss*,‡

University of Barcelona, Barcelona, Spain, and Bruker Daltonik GmbH, Fahrenheitstrasse 4, 28359 Bremen, Germany

Glycosylated proteins play important roles in a large number of biological processes. Therefore, a complete characterization in terms of glycan structures and glycoform heterogeneity is needed. In this paper, a combined approach based on glycan and intact glycoprotein analysis by capillary zone electrophoresis-electrospray-mass spectrometry (CZE-ESI-MS) is presented. Based on a new capillary coating, a CZE-ESI-MS method for the separation and characterization of intact glycoproteins has been developed and compared to a method recently introduced for the characterization of erythropoietin. The excellent glycoform separation results in high-quality mass spectra, high dynamic range, and good sensitivity, allowing the correct characterization of minor glycan modifications. Additionally, a CZE-ESI-MS separation method for underivatized N-glycans has been developed. The separation of glycans differing in the degree of sialic acids and repeats of noncharged carbohydrates is achieved. The separation power of the method is demonstrated by obtaining mobility differences in glycans differing only by 16 Da. A timeof-flight mass spectrometer allowed the correct identification of the glycan composition based on high mass accuracy and resolution, identifying even minor modifications such as the exchange of “O” by “NH”. An ion trap mass spectrometer provided structural information of the underivatized glycans from fragmentation spectra. The general applicability of both methods to glycoprotein analysis is illustrated for erythropoietin, fetuin, and r1acid glycoprotein. The results obtained by the glycan analysis allowed an unequivocal glyco-assignment to the masses obtained for the intact proteins as long as the protein backbone is well characterized. Furthermore, modifications found for intact proteins can be attributed to differences in the glycostructure. Glycosylation is one of the most common posttranslational modifications of proteins. The composition and structure of the attached carbohydrates play an important role in molecular * To whom correspondence should be addressed. E-mail: [email protected]. Fax: +49 421 2205 104. † University of Barcelona. ‡ Bruker Daltonik GmbH.

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recognition processes and biological activities of glycoproteins.1,2 The oligosaccharides can be linked to the protein backbone to a serine or threonine residue (O-glycosylation) or to an asparagine residue (N-glycosylation). The structures of N-linked glycans are of special analytical interest due to their size, heterogeneity, and variability. N-Linked glycans contain a common core with one or more antennas attached to the external mannose residues. The antennas usually consist of repeats of mannose or of one or more galactose-N-acetylglucosamine units (N-acetyllactosamine, LacNAc) that can be terminated in sialic acid units. In this sense, the primary structure of N-glycans is defined by number of antennas, sialylation degree, presence of fucoses, and number of LacNAc units. Therefore, the structural variability of the glycan residues, the number of glycosylation sites, and the different degree of site occupancy result in a mixture of glycoforms.2-4 There are several strategies for the study of glycoform heterogeneity in a certain glycoprotein: the direct analysis of intact glycoproteins, the analysis of glycopeptides obtained by tryptic digest, or the analysis of the released carbohydrates. Some interesting reviews about the different methods used for glycoprotein analysis and characterization have been published.2,5,6 On the intact glycoprotein level, nonspectrometric techniques as SDS-PAGE, lectin affinity chromatography, isoelectric focusing (also in a capillary), or capillary zone electrophoresis (CZE) are widely used. Especially, CZE has been successfully used in the separation of sialic acid isoforms of endogenous and recombinant glycoproteins,7-10 and it has proved its usefulness in clinical (1) Rudd, P. M.; Elliott, T.; Cresswell, P.; Wilson, I. A.; Dwek, R. A. Science 2001, 291, 2370. (2) Mechref, Y.; Novotny, V. Chem. Rev. 2002, 102, 321-369. (3) Kelly, J. F.; Locke, S. J.; Ramaley, L.; Thibault, P. J. Chromatogr., A 1996, 720, 409-427. (4) Harvey, D. J. Proteomics 2001, 1, 311-328. (5) Morelle, W.; Michalski, J. C. Curr. Anal. Chem. 2005, 1, 29-57. (6) Wuhrer, M.; Deelder, A. M.; Hokke, C. H. J. Chromatogr., B 2005, 825, 124-133. (7) Kinoshita, M.; Murakami, E.; Oda, Y.; Funakubo, T.; Kawakami, D.; Kakehi, K.; Kawasaki, N.; Marimoto, K.; Hayakawa, T. J. Chromatogr., A 2000, 866, 261-271. (8) Bristow, A.; Charton, E. Pharmaeuropa 1999, 11 (2), 290-300. (9) Sanz-Nebot, V.; Benavente, F.; Vallverdu´, A.; Guzman, N. A.; Barbosa, J. Anal. Chem. 2003, 75, 5220-5229. (10) Paca´kova, V.; Hubena´, S.; Ticha´, M.; Madera, M.; ×b3tulı´k, K. Electrophoresis 2001, 22, 459-463. 10.1021/ac060376g CCC: $33.50

© 2006 American Chemical Society Published on Web 06/29/2006

diagnosis and product quality assessment.11,12 In the past decade, mass spectrometric techniques have emerged as powerful tools for the analysis of large biomolecules.5 Electrospray ionizationmass spectrometry (ESI-MS) enables the precise mass determination of proteins.13 However, direct infusion of complex glycoproteins results in broad nonresolved signals as a consequence of an extremely high number of different glycoforms and potential salt adducts, all of them spread out over a charge distribution. Furthermore, the ionization efficiency in positive ESI is decreased, caused by the often negatively charged glycans.2,14 Therefore, prior to sensitive ESI-MS detection, the separation of the glycoforms is mandatory. CZE can be easily coupled to electrospray mass spectrometry; however, due to the nonstandard coupling technique, only a few papers for the separation and characterization of intact glycoforms by CZE-ESI-MS have been published.3,14-16 Successful CZE-ESIMS analysis of glycoproteins requires volatile background electrolytes at low pH as well as an appropriate capillary coating in order to increase both ionization efficiency and glycoform separation. Using this methodology, an intact molecular mass for every glycoform is obtained. However, the elucidation of the corresponding composition is difficult, since different carbohydrate combinations can lead to a certain mass.15 Thus, detailed characterization of the glycostructure is mandatory and complementary to intact protein characterization. The analysis of peptides as derived by “classical” enzymatic (e.g., tryptic) digestion of the protein also enables the characterization of the contained glycopeptides. This approach has some drawbacks: (i) there is an increase in sample complexity especially in those cases where the protein contains several N-glycosylation sites, (ii) glycopeptides with a large sugar fraction weakly retain on reversed-phase columns, and (iii) the ionization efficiency of the glycopeptides is lower both in ESI and in MALDI as compared to nonglycosylated peptides, resulting in weak sensitivity. The analysis of (enzymatically released) N-glycans is usually a simpler and faster method than glycopeptide analysis. Beside anion-exchange chromatography with pulsed amperometric detection, CZE with laser-induced fluorescent detection is frequently used to analyze glycans. This simple and sensitive method based on labeling with 8-amino-1,3,6-naphthalenetrisulfonate or 8-aminopyrene-1,3,6-trisulfonate (reductive amination) is a standard method for carbohydrate analysis and is commercially available. The few studies dealing with the analysis of N-glycans by CZE-ESI-MS used the same derivatization with chromophore or fluorophore groups, introducing also an ionic group needed for electrophoretic migration and an increase of ESI ionization.17-19 However, sialylation is a common modification found in complex glycans, remaining negatively charged, and (11) Ramdani, B.; Nuyens, V.; Codden, T.; Perpete, G.; Colicis, J.; Leanaerts, A.; Henry, J. P.; Legros, F. J. Clin. Chem. 2003, 49, 1854-1864. (12) Taverna, M.; Tran, N. T.; Merry, T.; Horvath, E.; Ferrier, D. Electrophoresis 1998, 19, 2572-2594. (13) Van den Heuvel, R. H. H.; Heck, A. J. R. Curr. Opin. Chem. Biol. 2004, 8, 519-526. (14) Demelbauer, U. M.; Plematl, A.; Kremser, L.; Allmaier, G.; Josic, D., Rizzi, A. Electrophoresis 2001, 25, 2026-2032. (15) Neusu ¨ ss, C.; Demelbauer, U.; Pelzing, M. Electrophoresis 2005, 26, 14421450. (16) Yeung, B.; Porter, T. J.; Vath, J. E. Anal. Chem. 1997, 69, 2510-2516. (17) Sandra, K.; van Beeumen, J.; Stals, I.; Sandra, P.; Claeyssens, M.; Devreese, B. Anal. Chem. 2004, 76, 5878-5886.

therefore, the glycans can be separated and detected in the negative ESI mode without a need for labeling.18 The correct characterization of complex mixtures of glycans can be done with high-accuracy mass measurements20 or by MS/ MS experiments.17,21 The study of the fragmentation pattern and the comparison with reference compounds and glycan libraries provide information about monosaccharide composition, branching, and linkages.4 In the current study, a combined approach based on the analysis of intact glycoproteins and underivatized glycans by CZEESI-MS is developed. A new general method for intact glycoprotein analysis is presented and shown to be generally applicable for several proteins such as bovine fetuin and bovine R1-acid glycoprotein. Excellent separation is achieved in this anodic CZE separation, and good mass resolution of TOF-MS allowed the correct characterization of comigrating and minor glycoforms. A method for the characterization of underivatized characterization of enzymatically released glycans of the complex type has been developed and is illustrated for the same set of glycoproteins. In this approach, the mass spectrometric detection was performed by two different mass analyzers. A TOF-MS allowed the exact mass determination of the glycans; an ion trap MS is used for glycan fragmentation. A different kind of information is provided by the glycan and intact glycoprotein approach, respectively. Both methods are discussed, and the complementary nature of the methods is demonstrated by combining the glycan information in order to elucidate an overall composition of the intact glycoprotein. EXPERIMENTAL SECTION Chemicals. 2-Propanol (Lichrosolv, HPLC gradient mode) and acetic acid (Suprasolve) were obtained from VW International (Darmstadt, Germany). Methanol (HPLC gradient grade) was obtained from J. T. Baker (Phillipsburg, NJ). Ammonia, 25%, was obtained from Merck (Darmstadt, Germany). Hexadimethrine bromide (Polybrene), PNGase F (proteomics grade), bovine fetuin, bovine R1-acid glycoprotein (bovine AGP), ammonium hydrogen carbonate, and 6-aminocaproic acid were supplied by Sigma/Fluka (Taufkirchen, Germany). UltraTol Dynamic Precoat LN was provided by Target Discovery (Palo Alto, CA). Trypsin Gold, mass spectrometry grade, was obtained from Promega (Madison, WI). Standard recombinant human erythropoietin (rHuEPO) was obtained as BRP from Pharmacopoeia (EDQM, European Pharmacopoeia, Council of Europe, Strasbourg, France). Deionized and organic-eliminated water was obtained using a Milli-Q water purification system (Millipore, Schwalbach, Germany). All solutions and background electrolytes were degassed by ultrasonication before use. Sample Treatment. For glycan analysis, a previous filtration procedure was needed for Pharmacopoeia rHuEPO due to the large amount of salts present in the formulation. A 50-µg sample of standard Pharmacopoeia rHuEPO was desalted and preconcentrated through a Microcon-10 cartridge (Millipore, Schwalbach, (18) Gennaro, L. A.; Delaney, J.; Vouros, P.; Harvey, D. J., Domon, B. Rapid Commun. Mass Spectrom. 2002, 16, 192-200. (19) Che, F.-Y.; Song, J.-F.; Zeng, R.; Wang, K.-Y.; Xia, Q.-C. J. Chromatogr., A 1999, 858 (2), 229-238. (20) Que, A. H.; Mechref, Y.; Huang, Y.; Taraszka, J. A.; Clemmer, D. E.; Novotny, M. V. Anal. Chem. 2003, 75, 1684-1690. (21) Wheeler, S. F.; Harvey, D. J. Anal. Chem. 2000, 72, 5027-5039.

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Germany). The filter membrane was initially washed with deionized water for 10 min at 14000g, and the sample was added to the filter and centrifuged at the same acceleration for 10 min. The residue was washed three times with a buffer containing 25 mM NH4HCO3, pH 8. The residue was recovered from the inverted cartridge by centrifugation for 3 min at 1000g, and this step was repeated by adding 10 µL of the same buffer to the membrane in order to increase the recovery. Glycans from rHuEPO, bovine fetuin, and bovine AGP were released by enzymatic deglycosylation with PNGase F. The procedure supplied by the manufacturer was modified slightly: 50 µg of the respective glycoprotein was dissolved into a final volume of 50 µL of 25 mM NH4HCO3, pH 8. Two units of PNGase F were added, and the reaction was carried out at 37 °C for 24 h. The reaction was stopped by heating for 5 min at 100 °C, and the sample was vacuum-evaporated down to 20 µL. For intact protein analysis, bovine AGP and bovine fetuin were dissolved in water at a final concentration of 5 µg/µL. CZE-ESI. CZE was performed on a Hewlett-Packard 3DCE (Agilent Technologies, Waldbronn, Germany). For CZE-MS coupling, a coaxial sheath-liquid sprayer was used (Agilent Technologies). Sheath liquid was delivered at 4 µL/min by a 5-mL gastight syringe (Hamilton, Reno, NV) using a Cole-Parmer syringe pump (Vernon Hill, IL). A nebulizer gas pressure of 0.2 mbar was applied to assist the spraying process. For intact glycoprotein analysis, separation was performed in fused-silica capillaries of 100 cm × 50 µm i.d. × 360 µm o.d. supplied by Polymicro Technologies (Phoenix, AZ). Capillaries were coated with Polybrene (PB) or UltraTol Dynamic Precoat LN (LN) in a procedure previously described.3 Briefly, new capillaries were activated with 1 M NaOH for 20 min, followed by 20 min of H2O. A solution containing 2% (w/v) PB or LN solution supplied by the manufacturer was flushed through the capillary for 40 min followed by 40 min of the respective background electrolytes (BGEs). In PB-coated capillaries, the BGE used was obtained adding 20% MeOH in an aqueous solution containing 1 M acetic acid, and the separation was carried out under a negative separation voltage of -30 kV. In LN-coated capillaries the BGE was 2 M HAc, and the separation was carried out under a positive voltage of +30 kV. In both systems, the sheath liquid contained 1% acetic acid in 2-propanol-water (1:1), and an electrospray potential of -4.5 kV was applied at the inlet of the MS (positive mode). For glycan analysis, the uncoated capillary measured 60 cm × 50 µm i.d. × 360 µm o.d. Different BGEs, with different amounts of MeOH and ammonia were tested (see later). The separation voltage was set to +30 kV at the inlet of the capillary. Injection was performed hydrodynamically at 50 mbar for 15 s. The sheath liquid consisted of 2-propanol-water (1:1). An electrospray potential of +4.5 kV was applied at the inlet of the MS (negative mode). MS. Glycan and intact protein analysis were performed using the micrOTOF (Bruker Daltonik, Bremen, Germany), an orthogonal accelerated time-of-flight mass spectrometer. For glycan analysis, the optimization of the transfer parameters and the MS calibration were performed with sodium formate cluster in the negative mode, whereas for intact protein analysis, the transfer parameters were optimized with TuneMix (Agilent Technologies) in order to obtain the best sensitivity at acceptable resolution 5386

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(12 000). The trigger time was set to 90 µs, corresponding to a mass range of 50-3000 m/z. Spectra were acquired by summarizing 20 000 single spectra, leading to a time resolution of 1.8 s. N-Glycan fragmentation of bovine fetuin was performed on a Bruker HCT, a nonlinear 3D ion trap mass spectrometer (Bruker Daltonik) in full scan or data-dependent MS2 scan modes. RESULTS AND DISCUSSION Intact Glycoprotein Analysis. The only studies successfully applying CZE-MS for glycoform separation of complex glycoproteins such as rHuEPO used the well-known dynamic coating Polybrene in order to reduce wall absorption and to provide a strong reversed EOF. In this sense, CZE separation and on-line detection by ESI-TOF-MS was successfully applied to the analysis of intact rHuEPO glycoforms.15,22 The determination of a high number of glycoforms with high mass accuracy allowed the differentiation of rHuEPO from different sources.15 Moreover, a clear differentiation was also obtained between epoetin-R and epoetin-β based on two ready-to-use drugs.22 These results proved the suitability of the method to characterize sugar heterogeneity in recombinant erythropoietin and to evaluate the quality of the product without any complicated sample treatment. To develop a general CZE-ESI-TOF-MS method for a broad range of complex glycoproteins, bovine fetuin and bovine AGP are used as model glycoproteins due to their known glycan complexity. Here, in a first step, the CZE-ESI-TOF-MS separation method based on the PB-coated capillary as previously applied to erythropoietin15,22 is also evaluated for its application to these two glycoproteins. Figure 1 shows the separation of the main sialoforms and the spectra obtained for bovine AGP. In Figure 1a. the separation is performed in a capillary coated with a PB solution using a BGE containing 1 M acetic acid and 20% MeOH. In these conditions, EOF is strongly reversed. The complex mixture of AGP glycoforms shows partial separation, and even glycoforms containing different sialic acid number (sialoforms) are not completely resolved. A more effective separation of the glycoforms is decisive to obtain better ESI response, to improve the quality of the mass spectra obtained, and to detect those isoforms present at low concentrations. To improve separation, a theoretical look on the resolution obtained in CZE is performed. The resolution of two peaks in CZE can be derived as follows:

R ∼ ∆uuˆ -1 ) (µ1,obs - µ2,obs)µobs-1 ) (µ1 - µ2)(µ - µEOF)-1

where R is resolution, u is electrophoretic velocity, µ is electrophoretic mobility, µobs is electrophoretic mobility as observed, and µEOF is electrophoretic mobility of EOF. The best resolution is obtained if the difference of the mobility of the analyte and the mobility of the EOF (µ - µEOF) is small. If the mobility of the ions as observed for the glycoproteins is small, a system with an EOF close to zero is expected to give better resolution compared to a system with high negative EOF (as it is given by the Polybrene coating). (22) Balaguer, E.; Neusu ¨ ss, C. Chromatographia, in press; DOI:10.1365/s10337006-0787-9.

Figure 1. (a) BPE and EIE of the main sialoforms of intact AGP, (b) mass spectrum obtained in the time interval between 20.6 and 20.7 min, and (c) deconvoluted spectrum. (a-c) Measured in a capillary 1 m × 50 µm, coated with Polybrene; BGE, 1 M acetic acid, 20% MeOH; separation voltage, -30 kV; (d) BPE and EIE of the main sialoforms of intact AGP, (f) mass spectrum obtained in the time interval between 19.9 and 20.0 min, and (g) deconvoluted spectrum. (d-f) Obtained in a capillary 1 m × 50 µm, coated with UltraTol Dynamic Precoat LN; BGE, 2 M acetic acid; separation voltage, +30 kV.

Thus, a coating providing a low EOF is tested in order to enhance mobility differences of glycoforms. A new acrylamidebased dynamic capillary coating, UltraTol Dynamic Precoat LN,23 decreases the EOF into a constant value at pH >4 and suppresses the EOF at a pH below this value. Using an acidic buffer containing 2 M acetic acid, the EOF is practically zero, the intact glycoforms are positively charged, and thus, they migrate to the cathode under a positive separation voltage. In Figure 1d, the results obtained using the LN coating are shown. The separation of the main sialoforms is considerably improved without compromising the migration times. Moreover, small shoulders appear in the BPE near the main sialoforms, corresponding to the partially overlapped signal of a glycoform with the same sialic acid number and containing one additional HexHexNAc. Panels b and e in Figure 1 show the mass spectra obtained for the major sialoform using a Polybrene and LN-coated capillaries, respectively. Panels c and f in Figure 1 show the charge-deconvoluted spectra. In these figures, the critical role of a good separation is revealed. LN coating provides improved glycoform separation, and thus, better (23) Chang, W. W.; Hobson, C.; Bomberger, D. C.; Schneider, L. V. Electrophoresis 2005, 26, 2179-2186.

quality mass spectra are obtained, leading to increased sensitivity. In both charge-deconvoluted spectra, a main peak for the major sialoform is observed together with a second less intense peak differing in ∼365 Da, which corresponds to the glycoform containing an additional HexHexNAc. Nevertheless, these peaks are considerably broad compared to the mass spectra obtained for erythropoietin15,22 and fetuin (see discussion below). Enlarging the deconvoluted spectrum this broadening can be attributed to heterogeneity on the level of ∼16 Da. Due to the improved glycoform separation, the resolution of this microheterogeneity is better defined in the case of the LN coating (Figure 1f.). The composition and structure causing this mass difference cannot be determined by the characterization of the intact glycoprotein. However, based on the subsequent glycan analysis (see below), it can be concluded that additional oxygen on the glycan is responsible for this repeated 16-Da distribution. Thus, these 16Da differences are very probably due to exchanges of acetylneuraminic acids (NeuAc) by glycolylneuraminic acids (NeuGc). This result, i.e., the distinction of isoforms differing by one oxygen each on the level of a 33-kDa protein, nicely shows the resolving power of the ESI-TOF MS. Analytical Chemistry, Vol. 78, No. 15, August 1, 2006

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Figure 2. (a) BPE and EIE obtained for intact bovine fetuin, (b) mass spectrum, and (c) deconvoluted spectrum obtained in a LNcoated capillary (cp. conditions in Figure 1).

Similarly, intact bovine fetuin has been analyzed. Up to now, no successful MS analysis for this widespread model glycoprotein has been achieved. Direct infusion analysis provides nonresolved mass spectra that cannot be properly deconvoluted. Thus, again separation is mandatory. Glycoform separation was performed using both Polybrene and LN-coated capillaries. Similarly to AGP and erythropoietin, better separation was obtained in the low-EOF capillary (LN coated). Figure 2shows the separation and the mass spectra obtained. As can be observed in Figure 2a, only partial glycoform separation is observed in the BPE; however, taking the selectivity of the attributed mass, a separation is clearly obvious. Panels b and c in Figure 2 show the mass spectrum and the charge-deconvoluted spectrum in the interval 19.7-20.0 min, respectively. The spectra obtained here present narrower peaks than the spectra obtained for bovine AGP, because no heterogeneity on the level of 16 Da (or similar) is observed for fetuin. However, several signals corresponding to different glycoforms that contain different number of NeuAc and HexHexNAc comigrate in the time interval chosen. 5388

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Summarizing the presented method for intact glycoprotein analysis, it can be stated that a simple and fast method has been developed to obtain an overall picture of the composition of glycoproteins by achieving extraordinary separation of the different glycoforms. The approach is expected to improve the ability to characterize various glycoproteins both in biotechnological development and in QC of pharmaceutical production. Glycan Analysis. Structural information provided by the intact glycoform analysis is limited; i.e., the exact composition in type of glycans belonging to a certain intact glycoform cannot be unequivocally predicted, because several monosaccharide combinations lead to a certain mass. Thus, prior information about the type and amount of glycans present in the studied glycoproteins must be known before a complete carbohydrate composition can be assigned to every observed intact glycoform. Furthermore, modifications observed on the glycoprotein level (e.g., acetylation or oxidation) can be assigned either to the protein backbone or the glycostructure when analyzing the glycans. (1) CZE-MS Separation. The analysis of N-linked oligosaccharides of the complex type has been optimized using three different glycoproteins. Bovine fetuin and bovine AGP have endogenous origins, whereas human erythropoietin is obtained from recombinant technology. These glycoproteins are known to contain different types of complex N-glycans with different degrees of terminal sialic acids. These acidic analytes are expected to be detected in negative ESI mode, which indeed turns out to be superior to positive ESI detection. The MS analysis by direct infusion of the PNGase F-released N-glycans results in complex spectra. Their interpretation is difficult, and sometimes minor glycans cannot be successfully detected.18 Therefore, previous LC or CE separations are mandatory for a complete glycan characterization. Here, a straightforward CZE-ESI-MS separation method for nonderivatized glycans detected in negative ionization mode has been developed. At first some typical MS-compatible background electrolytes based on ammonium acetate, ammonium formate, and ammonium carbonate were tested. These systems did not provide any successful separation of the different N-glycans at reasonable peak shape. An improvement in separation was obtained when a background electrolyte based on 6-aminocaproic acid was applied.18 Different percentages of MeOH and different concentrations of NH3 were evaluated in order to improve the separation. By increasing the MeOH content, the migration times of each glycan increase and the peaks become broader. In contrast, adding higher amount of NH3, the migration times decrease, the peaks become sharper, and signal intensity is higher. Finally, the best resolution and intensity are obtained in a background electrolyte made by mixing an aqueous solution containing 100 mM 6aminocaproic acid and 0.9 M NH3 with MeOH in a ratio of 30:70. Figure 3 shows the separation obtained for the main N-glycans released from bovine fetuin, bovine AGP, and rHuEPO as detected by the ESI-TOF MS. The separation is carried out under normal polarity and works for all the different N-glycan mixtures released from the three different glycoproteins studied. Increasing the sialic acid number of the glycans, the negative charge of the glycan is increased and the glycan has higher mobility against the EOF. Thus, glycans containing a higher amount of sialic acids appear later in the electropherogram. Increasing the size of the negative

Figure 3. Extracted ion electrophreograms obtained for the main glycans released from bovine fetuin (a), bovine AGP (b), and Pharmacopoeia rHuEPO (c). Conditions: 60 cm × 50 µm capillary; BGE, 100 mM caproic acid, 0.9 N NH3 + 70% MeOH; injection, 15 s 50 mbar; separation voltage, +30 kV; sheath liquid 2-propanol-water 1:1 delivered at 4 µL/min.

charged glycan (adding antennas, HexHexNAc units, or fucose units), the mobility is lower, and thus, heavier glycans are detected earlier. This is nicely illustrated by the perfect separation of the erythropoietin glycans containing four antennas (Ant) and four sialic acids (SiA) from its isoform containing an additional LacNAc repeat as can be seen in the two last peaks of Figure 3c. In this way, the main glycans are successfully separated, and just some minor glycans are partially overlapping; however, they are easily distinguished in the mass spectra. As an example, the separation of several fucosylated from the respective nonfucosylated glycans is shown in Figure 3a and b for bovine fetuin and

AGP, respectively. The respective fucosylated peaks appear slightly earlier, though not baseline resolved. In Figure 3b, the released glycans from bovine fetuin are displayed. Peaks corresponding to the triantennary glycans containing three and four sialic acids are surprisingly broad. The reason for this might be that triantennary glycans can present several isomers corresponding to different types of branching, and they can be slightly separated by CZE due to the different apparent size of each one. Biantennary glycans do no present broad peaks because different isomers due to different kinds of branching are not possible. Figure 4 demonstrates in more detail the separation power of the technique. The averaged mass spectrum for the biantennary glycan with three sialic acids for bovine AGP is shown in Figure 4a. The glycans typically distribute over a charge envelope; in this case, doubly and triply charged peaks appear (for glycans with 4 SiA also quaterly charged glycans can be observed). The detailed view of the triply charged peaks is shown in Figure 4b. Clearly the presence of a set of four triply charged peaks differing by 16 Da can be observed. The first peak corresponds to a glycan containing three NeuAc, the second peak contains two NeuAc and one NeuGc (it differs from a NeuAc in one O), the third peak contains one NeuAc and two NeuGc, and finally, the fourth peak corresponds to a glycan containing three NeuGc. Obviously the given structure is not necessarily the only one possible for the found composition; however, for clarity reasons this most probable structure is assumed, in accordance with the literature.24,25 The separation of these four isoforms of the biantennary glycan with three sialic acids is shown in Figure 4d. Mobility differences are clearly observed depending on the degree of NeuGc substitution. Though no baseline separation is achieved, these detected mobility differences show the excellent separation power of the technique. The method allows the detection of mobility differences provided by only 16 Da in high molecular mass substances (2000-3000 Da) in a separation carried out in less than 15 min. Table 1 lists the main glycans observed for bovine fetuin and bovine AGP with the respective areas obtained. As can be observed in Figure 3c, for rHuEPO, the main glycans were tetraantennary with four sialic acids, and they presented several HexHexNAc (i.e., N-acetyllactosamine) repetitions. For the two natural glycoproteins, fetuin and AGP, the main glycans were partially fucosylated, whereas for rHuEPO, a recombinant glycoprotein, all the observed glycans were fucosylated. The latter is in agreement with the results of the analysis of intact rHuEPO, where a mass difference of 149 Da was never observed. All the different types of glycans found for these three glycoproteins agree to a large extent with the results obtained by other authors in glycopeptide and glycan analysis.18,24,25 Moreover, even for these model glycoproteins, we found minor glycans to our knowledge not described in the literature, e.g., all the fucosylated glycans for fetuin and AGP, the tetraantennary glycan with three sialic acids for fetuin, the tetraantennary glycan with three sialic acids, and the triantennary glycan with two sialic acids for AGP. Therefore, this method is demonstrated to be an excellent tool for glycan analysis. (24) Nakano, M.; Kakehi, K.; Tsai, M. H.; Lee, Y. C. Glycobiology 2004, 14 (5), 431-441. (25) Rush, R. S.; Derby, P. L.; Smith, D. M.; Merry, C.; Rogers, G.; Rohde, M. F.; Katta, V. Anal. Chem. 1995, 67, 1442-1452

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Table 1. Main Glycans Obtained for Bovine Fetuin and Bovine AGP carbohydrate structure

Figure 4. (a) Overall mass spectrum obtained in the time interval 12.4-12.7 min. (b) Extended mass spectrum between 836 and 855 m/z. (c) Deconvoluted mass spectrum of 2Ant 3NeuAc glycan. (a) EIE of trisialic, biantennary glycans of bovine AGP with different NeuAc and NeuGc proportion.

(2) Mass Accuracy. Mass accuracy, i.e., the deviation of the measured monoisotopic mass from the theoretical mass, and the isotopic resolution, are important features in MS analysis. Concerning these two terms, orthogonal acceleration time-of-flight 5390 Analytical Chemistry, Vol. 78, No. 15, August 1, 2006

theo mass

calc mass

err (ppm)

area 244 334 2 91 139 439 887 282 048 67 876 126 573 48 266 503 926 888 2 129 897 109 818 9 15 941 61 102 14 386 144 147

2Ant 2SiA 2Ant 2SiA 1Fuc 2Ant 3SiA 3Ant 2SiA 3Ant 2SiA 1Fuc 3Ant 3SiA 3Ant 3SiA 1Fuc 3Ant 4SiA 3Ant 4SiA 1Fuc 3Ant 5SiA 3Ant 5SiA 1Fuc 4Ant 3SiA 4Ant 3SiA 1Fuc 4Ant 5SiA

Fetuin 2221.7757 2221.7658 2367.8336 2367.8304 2512.8711 2512.8715 2586.9079 2586.9024 2367.8336 2732.9624 2878.0033 2878.0030 3024.0612 3024.0624 3169.0987 3169.0937 3315.1567 3315.1508 3460.1942 3460.1881 3606.2521 3606.2460 3243.1355 3243.1268 3389.1934 3389.1753 3825.3264 3825.3220

-4.5 -1.4 0.1 -2.1 -1.3 -0.1 0.4 -1.6 -1.8 -1.8 -1.7 -2.7 -5.4 -1.1

2 Ant 1NeuAc 2 Ant 2NeuAc 2 Ant 2NeuAc 1Fuc 2 Ant 3NeuAc 2 Ant 3NeuAc 1Fuc 2 Ant 4NeuAc 3 Ant 2NeuAc 3 Ant 3NeuAc 3 Ant 4NeuAc 4 Ant 3NeuAc

R1-Glycoprotein 1930.6803 1930.6948 2221.7757 2221.7793 2367.8336 2367.8416 2512.8711 2512.8845 2658.9290 2658.9405 2803.9666 2803.9757 2586.9079 2586.9204 2878.0033 2878.0133 3169.0987 3169.1005 3243.1355 3243.1522

7.5 1.6 3.4 5.3 4.3 3.3 4.8 3.5 0.6 5.1

4 343 202 200 6 510 408 356 14 386 92 799 4 766 53 733 21 514 11 029

(oaTOF) analyzers are the detectors of choice. Furthermore, high sensitivity, broad m/z range, and a high acquisition rate make oaTOF-MS a suitable instrument for coupling with such highefficiency separation techniques as CZE.26 Table 1 summarizes the theoretical and observed molecular masses and the respective mass accuracy obtained for bovine fetuin and bovine AGP. High mass accuracy better than (5 ppm allows the elucidation of the glycan composition with high confidence. For bovine fetuin, excellent mass accuracy is obtained here (few ppm), similar to values obtained in previous studies applying FTICR-MS.21 In most cases, the measured mass of a glycan lead to a single glycan composition because the number and type of monosaccharides are limited. Only the structures containing NeuAc are displayed for AGP, and the other structures containing one to four NeuGc (depending on the number of sialic acids) are omitted. In terms of mass accuracy, all AGP glycans seem to have positive deviation, slightly higher than the deviation obtained for fetuin. For the 2Ant 3NeuAc glycan, the theoretical mass (Table 1) corresponds to the second peak appearing in Figure 4c (2512.8845 Da). The first small peak at 2511.8959 Da does not belong to the isotopic pattern of this compound, but it is rather a signal of another substance with a molecular mass ∼1 Da lower. The most probable modification is an O T NH exchange, which leads to a glycan with a mass of 0.984 Da less. This theoretical value is in agreement with the observed distance between the first two peaks in the mass spectrum. Moreover, no other simple combination of element exchange leads to a similar difference. The positive deviation on mass accuracy as given in Table 1 for glycans from bovine AGP are caused by the overlapping of the second isotope of the NH(26) Guilhaus, M.; Selby, D.; Mlynskl, V. Mass Spectrom. Rev. 2000, 19, 65107.

Figure 5. MS2 obtained for a trisialic, triantennary glycan released from bovine fetuin (a) in negative and (b) in positive mode.

containing glycan with the first isotope of the O-containing glycan, provoking a slight displacement to higher molecular masses for the main compound. These results prove that the high mass accuracy in combination with the determination of the correct isotopic pattern allows the characterization of two glycans that differ from each other by 1 Da. Since the isotopic ratio is correctly measured by this instrument, a quantitative ratio can be calculated, assuming the same response in the electrospray. About 16% of the glycan is converted to an aminoglycan. Therefore, CZE glycan separation in combination with highaccuracy MS analysis allows on one hand the correct characterization of glycans over a wide dynamic range and, on the other hand, the quantification of the glycans present in a certain glycoprotein. (3) Glycan Fragmentation. The previous described results obtained by an ESI-TOF MS lead to a composition of the glycans. To evaluate the possibility to obtain structural information, online tandem mass spectrometry has been applied to the CEseparated glycans. Fragmentation studies of fetuin glycans were performed on-line in an ion trap mass spectrometer. Different types of fragmentation are possible: loss of the adduct (i.e., - H2O), glycosidic cleavages that result from breaking the bond between two sugar rings, and cross-ring cleavages. In general, glycosidic cleavages provide information on sequence and branching, whereas cross-ring cleavages provide information about the

linkages.4 Glycan identification by tandem mass spectrometry can be performed using a glycan database such as Glycosuite, where the monoisotopic masses of fragments are compared to theoretical fragments generated from oligosaccharides in the database.27 Figure 5 shows MS2 spectra from a parent ion corresponding to an underivatized triantennary trisialylated glycan from fetuin. Figure 5a shows the fragmentation obtained in negative ESI mode, in the experimental conditions described in above. The most common fragments in this mode are the sialic acid loss and fragments produced by cross-ring cleavages. Glycans with several sialic acid residues usually gave intense fragment ions from the loss of each individual sialic acid because of the extreme lability of this residue. This minimizes the capability to generate fragments containing structural information. Moreover, in negative mode, cross-ring cleavages are favored, and due to the branching and the presence of several sialic acids that give negative charges to the generated fragments, several pathways for fragmentation are observed, making the interpretation of the generated MS2 spectra more complicated.4,28 (27) Joshi, H. J.; Harrison, M. J.; Schulz, B. L.; Cooper, C. A.; Packer, N. H.; Karlsson, N. G. Proteomics 2004, 4, 1650-1664. (28) Karlsson, N. G.; Wilson, N. L.; Wirth, H. J.; Dawes, P.; Joshi, H.; Packer, N. H. Rapid Commun. Mass Spectrom. 2004, 18, 2282-2292.

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Table 2. Main Intact Glycoforms for Bovine AGP 25Hex20HexNAc

9 SiA 4NeuAc-5NeuGc 10 SiA 5NeuAc-5NeuGc 11 SiA 5NeuAc-6NeuGc 12 SiA 6NeuAc-6NeuGc 13 SiA 6NeuAc-7NeuGc 14 SiA 7NeuAc-7NeuGc 15 SiA 7NeuAc-8NeuGc 16 SiA 8NeuAc-8NeuGc

26Hex21HexNAc

measured

calculated

measured

calculated

32169.8

32170.3

32531.6

32535.6

32461.2

32461.5

32825.4

32767.3

32768.8

33059.7

27Hex22HexNAc measured

calculated

32826.9

33189.9

33192.2

33134.0

33134.1

33497.6

33499.5

33060.0

33423.4

33425.4

33789.0

33790.7

33366.9

33367.3

33731.7

33732.6

34095.4

34098.0

33657.7

33658.6

34034.4

34023.9

34388.9

34389.2

33965.1

33965.8

34331.6

34331.1

34694.2

34696.5

34255.4

34257.1

Reasonable detection in positive ESI is not possible with the aminocaproic acid-based system. Therefore, a BGE containing 25 mM NH4Ac and 10 mM NH3, and a sheath liquid containing 0.5% formic acid in 50:50 2-propanol-water are used to obtain some MS/MS spectra in positive mode in order to compare MS/MS fragmentation in positive and negative ESI mode. The strongly acidic conditions in the sheath liquid are necessary to obtain a suitable positive ionization, which otherwise is impeded by the sialic acids. Nevertheless, a weak separation is achieved, and sufficient signal intensity is achieved in order to obtain some spectra for the most abundant glycans in the positive ESI mode. Figure 5b shows the fragmentation spectrum carried out in positive ESI mode again for the underivatized triantennary trisialylated glycan from fetuin. It can be noticed that most fragments appearing in the positive mode are products of glycosidic cleavages (Hex 162 Da, HexNAc 203 Da, NeuAc 291 Da). However, no differentiation could be obtained between the different possible isomers in terms of branching and type of linkages. Nonderivatized fetuin glycans were analyzed previously by CZE-ESI-MS18 in a similar separation method based on aminocaproic acid BGE. In MS/MS mode, facile loss of sialic acids was observed; thus, little useful information about the glycan structure was obtained. So far, most of the glycan analyses have been performed after derivatization (methylation) in order to give access to glycans by classical reversed-phase liquid chromatography.4,30 More effort certainly needs to be put into the interpretation of MS/MS spectra of underivatized glycans; however, this is beyond the scope of this paper and needs the extensive characterization of standards. Glycan and Intact Glycoprotein Combined Data. The presented method for glycan analysis allows the characterization of carbohydrates of the complex type in great detail; i.e., modifications down to NH-O exchange can be studied. However, this method alone does not reveal any total composition of the (29) Suzuki, H.; Mu ¨ ller, O.; Guttman, A.; Karger, B. L., Anal. Chem. 1997, 69 (22), 4554-4559. (30) Weiskopf, A. S.; Vouros, P.; Harvey, D. J. Anal. Chem. 1998, 70, 44414447

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glycoprotein. Contrariwise, the analysis of intact glycoproteins (as described under Intact Glycoprotein Analysis) does provide an overall composition, but is limited in the elucidation of the composition of the glycoprotein, since several monosaccharide combinations can lead to a certain molecular mass. Combining both methods, an overall carbohydrate composition of the intact protein can be given. That is, a glycan composition can be assigned to every molecular mass obtained for each intact glycoform (cp. Intact Glycoprotein Analysis) by taking into account the results obtained by the analysis of glycans (type and amount of each glycan present in the studied glycoprotein). For the correct glycoassigment, the molecular mass of the protein backbone must be exactly known. However, the protein backbone of the bovine AGP has not been extensively studied. Taking the molecular mass of the amino acid sequence, no suitable glycan combination leads to the observed molecular masses. Therefore, some modifications of the protein backbone mainly based on the modifications present in the human AGP are supposed, such as the pyro-Glu in the N-terminal amino acid, the presence of two disulfide bonds, and the loss of two hydroxyl groups, obtaining a protein mass of 21 352 Da. Bovine AGP presents five N-glycosylation sites. The analysis of the glycans revealed that biantennary glycans with two and three sialic acids are the most abundant glycans. Therefore, the first main peak observed in the BPE obtained in Figure 1d probably contains 10 sialic acids, assuming all the N-glycosylation sites contain a disialic glycan. Consistently, the following main peaks contain 11, 12, 13, and 14 sialic acids, as is annotated in the figure. Thus, the glycoform shown in Figure 1f (33 059.7 Da) contains two biantennary glycans with three sialic acids and three biantennary glycans with two sialic acids. Hence, the total monosaccharide composition of this glycoform probably consists of 25 Hex, 20 HexNAc, 6 NeuAc, and 6 NeuGc. Added to the protein backbone, this leads to a theoretical mass of 33 060.0 Da which is in agreement with the experimental mass observed. Table 2 lists all the main glycoforms observed in the intact glycoform approach for bovine AGP and their respective carbohydrate composition. The mass consistency of the technique on the level of nonisotopically resolved masses is obvious: Glycoforms with higher

intensity, i.e., those that are only composed of biantennary glycans (25Hex20HexNAc), show small differences (∼1 Da) between the calculated and the observed molecular masses. Glycoproteins with higher HexHexNAc numbers presented higher errors because their intensity is lower, the microheterogeneity of NeuAc and NeuGc is less defined, and, moreover, some mass spectra of other sialoforms are overlapped. Bovine fetuin is known to contain three O-glycosylation sites and three N-glycosylation sites. O-Glycans cannot be released without any endoglycosidase as PNGase F. Therefore, the results obtained in the glycan analysis, the molecular mass of the protein backbone, and the studies performed by other authors for the characterization of the O-glycans were taken into account for fetuin in order to perform a correct carbohydrate assignment to every intact glycoform observed. O-Glycans from bovine fetuin are usually composed of a few Hex and HexNAc units and one or two sialic acids.31 From the study performed in Intact Glycoprotein Analysis, the three N-glycosylation sites of bovine fetuin are mainly occupied by bi- and triantennary glycans with two, three, and four sialic acids. In this sense, a probable carbohydrate composition can be calculated for every intact glycoform. Thus, a probable composition for the glycoform displayed in Figure 2 with a molecular mass of 47 188.8 consists of the protein backbone (36 340.8 Da), one triantennary glycan with four sialic acids and two triantennary glycans with three sialic acids from the Nglycosylation sites, and three NeuAcHexHexNAc from the Oglycosylation sites. The global carbohydrate composition for this glycoform contains 21 hexoses, 18 HexNAc, and 13 sialic acids (Mr,av 47 189.6 Da). Moreover, intact glycoform analysis allows the partial characterization of glycoprotein heterogeneities related to the occupancy degree of the glycosylation sites (“macroheteroneneity”), whereas glycan analysis only can provide information related to the type and amount of the released N-glycans. This is demonstrated in the intact bovine fetiun analysis. In the obtained BPE (Figure 2), a first set of glycoforms of lower molecular mass is observed that cannot be correctly assigned with the six described glycosylation sites and the glycan structures found in the glycan analysis. Nevertheless, differences between the two sets of glycoforms can be attributed to a complete glycan. As shown in Figure 2a, the mass 47 188.8 differed from the glycoform of 44 037.5 Da by 3151.3 Da; this corresponds exactly to a triantennary glycan with four sialic acids. In another example, the difference between the glycoform of 46 531.7 Da and the glycoform of 43 670.5 Da is 2861.2 Da, which corresponds to a trisialic triantennary glycan. These results are only explained by the existence of a free (31) Royle, L.; Mattu, T. S.; Hart, E.; Langridge, J. I.; Merry, A. H., Murphy, N.; Harvey, D. J.; Dwek, R. A.; Rudd, P. M. Anal. Biochem. 2002, 304, 70-90. (32) Kolch, W.; Neusu ¨ss, C.; Pelzing, M.; Mischak, H. Mass Spectrom. Rev. 2005, 24, 959-977.

N-glycosylation site in the set of glycoforms appearing at earlier migration times. CONCLUSIONS The presented intact glycoprotein analysis by CZE-ESI-MS is a fast method that does not require any complicated sample treatment. An overall composition for a wide range of glycoforms can be given and their relative intensity evaluated. An efficient separation of the intact glycoforms is presented, enabling us to decrease mass spectra complexity and resolve correctly small modifications as oxidations or acetylations. Moreover, this approach allows the characterization of the occupancy degree of the glycosylations sites (macroheterogeneity), though no site-specificity can be given. The method is expected to be of general applicability for various complex glycoproteins. Some first tests on the stability and reproducibility of the coating are promising. Additionally, a fast and simple method by CZE-ESI-MS of released N-glycans from the same set of glycoproteins is presented. The glycan method provides detailed information about the type and composition of glycans, i.e., the antenna number, presence of fucosyl units, or the degree of sialylation of each single glycan. Moreover, small modifications as NeuAc-NeuGc exchanges are better resolved in the glycan range than in the intact glycoprotein analysis. The combination of both methods reveals first a clear attribution of modifications observed for the intact glycoprotein to the carbohydrate part of the molecule. Second, an overall composition can be given as far as the protein backbone is well characterized. Key application of the method consist of in-depth characterization of isolated proteins, which are available in low-microgram amounts. Thus, the presented method may also assist to resolve some biological aspects about natural glycoproteins. The direct analysis of serum by CE-MS already leads to the detection of several glycoproteins.32 Thus, it is expected that the presented approach is an appropriate tool for the characterization of the glycosylation pattern of high-to-medium abundant glycoproteins in serum after appropriate preconcentration and precleaning procedures. ACKNOWLEDGMENT We thank Will Chang (Target Discovery) for the gift of the coating material.

Received for review February 28, 2006. Accepted May 22, 2006. AC060376G

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