Anal. Chem. 2001, 73, 4755-4762
Accelerated Articles
Qualitative and Quantitative Analysis of the Glycosylation Pattern of Recombinant Proteins N. Viseux, X. Hronowski, J. Delaney, and B. Domon*,†
Biogen Inc., Cambridge, Massachusetts 02142
Over the past decade, the growing number of recombinant glycoproteins used as therapeutic agents has prompted the development of robust and rugged methodologies for characterizing the glycosylation pattern of such molecules. The present study describes an alternative to the widely used HPLC approaches for profiling the N-glycan heterogeneity of proteins. The method encompasses the enzymatic deglycosylation of the glycoprotein, the permethylation of the released oligosaccharides, and the subsequent analysis of these derivatives by either matrixassisted laser desorption/ionization or electrospray mass spectrometry. This methodology showed excellent correlation when compared with results obtained by an orthogonal technique such as the HPLC of 2-aminobenzamide-labeled glycans. In addition, it gives a more detailed insight into the glycosylation pattern by unambiguously identifying and quantifying the various glycoforms present in the mixture. Despite a somewhat complex sample preparation, reproducibility and robustness of the method were excellent. In the case of very heterogeneous glycan pools, simplification of the glycosylation pattern was achieved by performing enzymatic desialylation prior to deglycosylation and derivatization, leading to a more direct determination of the antennary distribution as well as the identification of minor components.
The use of recombinant proteins as therapeutic agents has dramatically increased over the past decade.1 The characterization of recombinant glycoproteins constitutes a challenge as they usually exhibit extensive sample heterogeneity. The analysis of such products is essential at all stages of the invention process. * Corresponding author. Tel.: (240) 453-3210; fax: (240) 453-4112; e-mail:
[email protected]. † Current address: Celera Genomics, 45 West Gude Dr., Rockville, MD 20850. (1) Drews, J. Science 2000, 287, 1960-1964. 10.1021/ac015560a CCC: $20.00 Published on Web 09/19/2001
© 2001 American Chemical Society
It starts at the first expression of a new glycoprotein in order to identify the glycosylation pattern and continues all through the development and optimization of the production process to detect changes. As a uniform glycosylation favors the constant biological activity of the product, monitoring batch-to-batch consistency is necessary. In contrast to proteins and nucleic acids, there is not yet a universal methodology for oligosaccharide analysis. Thus, the development of robust and rugged methods to carry out the characterization of glycosylation profiles has become a challenge for the analytical chemist. The biosynthesis of N-linked glycans follows a complex, enzymatically controlled pathway. As it is a well-defined process, only a small subset of all theoretical structures is actually synthesized, therefore tremendously reducing their diversity and simplifying their characterization. The primary structure of Nglycans is defined by a set of critical features that includes (i) the number of antennae, (ii) the degree of sialylation, (iii) the presence of fucosyl residues (on the core and on the antennae), and (iv) the number of lactosamine units. In most cases, the monosaccharide composition, which consists of a very limited set of building residues (galactose (Gal), mannose (Man), fucose (Fuc), N-acetylglucosamine (GlcNAc), N-acetylneuraminic acid (NeuAc)), can be directly deduced from the molecular mass of the oligosaccharide. In this way, the identification of the various glycoforms observed in a mass spectrum is fairly straightforward. To differentiate unresolved isobaric oligosaccharides, additional structural information is required to alleviate ambiguities that may exist on linkages, such as Gal(1-3)GlcNAc versus Gal(1-4)GlcNAc, NeuAc(2-3)Gal versus NeuAc(2-6)Gal, and (2,4)- versus (2,6)disubstituted mannose residues. Such information can be derived from methylation analysis,2 tandem mass spectrometry experiments,3-5 or the combination of exoglycosidase digestions and mass spectrometry.6 (2) Hellerqvist, C. G. Methods Enzymol. 1990, 193, 554-573. (3) Gillece-Castro, B. E.; Burlingame, A. L. Methods Enzymol. 1990, 193, 689712.
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Several approaches have been proposed to analyze the distribution of N-glycans released from proteins either enzymatically or chemically (e.g., PNGase F treatment or hydrazinolysis). Those include chromatographic and electrophoretic methods as well as mass spectrometry. In the former case, the released oligosaccharide mixtures are reductively aminated with a fluorophore to ensure proper detection. The most commonly used derivatizing agents are 2-aminobenzamide (2AB),7 2-aminobenzoic acid (2AA),7,8 and 2-aminopyridine (2AP).9 The modified oligosaccharides are most often analyzed by ion exchange chromatography. Similarly, electrophoresis of glycans derivatized with, for instance, 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) was reported.10,11 In addition, separation of native oligosaccharides by high-pH anion exchange chromatography in conjunction with pulsed amperometric detection is another widely used technique.7,12 Several mass spectrometry alternatives have been reported for carbohydrate analysis, using electrospray (ES) or matrix-assisted laser desorption/ionization (MALDI) for analysis of free or derivatized oligosaccharides.13-15 More specifically, it was demonstrated that permethylation can significantly enhance the mass spectrometric response of carbohydrates in both positive and negative modes. In addition pertinent structural information can be derived.5,16-18 The aim of the present study was to establish and validate a robust mass spectrometric method for characterizing the glycosylation pattern of recombinant proteins that allows a rapid identification of the various glycoforms as well as their (semi)quantitation. The approach encompasses the enzymatic deglycosylation with PNGase F, the permethylation of the glycan pool, and the analysis of these derivatives by MALDI or electrospray mass spectrometry. To validate the method, the results were compared with those obtained by an orthogonal technique, namely, the anion exchange chromatography of 2-aminobenzamide-labeled oligosaccharides. EXPERIMENTAL SECTION The R1-acid glycoprotein was purchased from Sigma (St. Louis, MO). The recombinant soluble CD4 glycoprotein was produced at Biogen Inc. The enzymes PNGase F and recombinant neuraminidases NANase I and NANase III cloned from Streptococcus pneumoniae and Arthrobacter ureafaciens, respectively, were purchased from Glyko Inc. (Novato, CA). The Signal 2-AB labeling (4) Weiskopf, A. S.; Vouros, P.; Harvey, D. J. Anal. Chem. 1998, 70, 44414447. (5) Viseux, N.; De Hoffmann, E.; Domon, B. Anal. Chem. 1998, 70, 49514959. (6) Geyer, H.; Schmitt, S.; Wuhrer, M.; Geyer, R. Anal. Chem. 1999, 71, 476482. (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) Anumula, K. R. Anal. Biochem. 2000, 283, 17-26. (9) Hase, S. Methods Enzymol. 1994, 230, 225-237. (10) Jackson, P. Methods Enzymol. 1994, 230, 250-265. (11) Raju, T. S. Anal. Biochem. 2000, 283, 125-132. (12) Hardy, M. R.; Townsend, R. R. Methods Enzymol. 1994, 230, 208-225. (13) Rouse, J. C.; Strang, A. M.; Yu, W.; Vath, J. E. Anal. Biochem. 1998, 256, 33-46. (14) Harvey, D. J. Mass Spectrom. Rev. 1999, 18, 349-450. (15) Harvey, D. J. J. Mass Spectrom. 2000, 35, 1178-1190. (b) Harvey, D. J. J. Am. Soc. Mass Spectrom. 2000, 11, 900-915. (16) Sheeley, D. M.; Reinhold, V. N. Anal. Chem. 1998, 70, 3053-3059. (17) Reinhold, B. B.; Chan, S. Y.; Reuber, T. L.; Marra, A.; Walker, G. C.; Reinhold, V. N. J. Bacteriol. 1994, 176, 1997-2002. (18) Linsley, K. B.; Chan, S. Y.; Chan, S.; Reinhold, B. B.; Lisi, P. J.; Reinhold, V. N. Anal. Biochem. 1994, 219, 207-217.
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kit (K-404), the GlycoSep C column (I-4721), and GlycoClean S cartridges (I-4726) were obtained from Glyko Inc. The C8 SepPak cartridges were bought from Waters (Milford, MA). Sodium hydroxide and chloroform were obtained from Aldrich (Milwaukee, WI); dimethyl sulfoxide and iodomethane were from Pierce (Rockford, IL) and Fluka (Milwaukee, WI), respectively. The R-cyanohydroxycinnamic acid matrix for MALDI MS was bought from Aldrich, and the HPLC solvents were purchased from J.T. Baker (Philipsburg, NJ). The HPLC analyses were performed on a 2690 Alliance system equipped with a 474 fluorescence detector (Waters). The electrospray mass spectrometry measurements were carried out on a Quattro II instrument equipped with a Z-spray ion source (Micromass, Manchester, U.K.). The MALDI MS measurements were carried out on a Voyager DE-STR time-of-flight instrument (Applied Biosystems, Framingham, MA) equipped with a nitrogen laser with an emission wavelength at 337 nm. Preparation of the Glycan Pools. The glycoprotein (typically 1-2 nmol) was denatured in 0.5% SDS and 1% β-mercaptoethanol (100 °C, 5 min) and deglycosylated by enzymatic digestion overnight with PNGase F at 37 °C in 25 mM sodium phosphate (pH 7.5). The glycan pool was separated from the protein by ethanol precipitation, aliquoted in two equal portions, and evaporated to dryness. Enzymatic Desialylation of Glycoproteins. The native glycoprotein (1 nmol) was desialylated using either the recombinant neuraminidase NANase I or NANase III at 37 °C overnight according to the manufacturer’s recommendations, namely, in 50 mM sodium phosphate (pH 6.0) with 10 munits of enzyme. The resulting product was then evaporated to dryness and submitted to deglycosylation. HPLC Separation of 2-Aminobenzamide-Derivatized NGlycans. An aliquot of the glycan pool (1 nmol) was reductively aminated with 2-aminobenzamide using the Signal 2-AB labeling kit. The excess of reagents was removed with a GlycoClean S cartridge, and the derivatized oligosaccharides were concentrated prior to HPLC analysis. The 2AB-labeled glycan mixture was separated on a GlycoSep C column (0.4 mL/min) using an acetonitrile/water/ammonium acetate gradient. In a first step, the water proportion was increased from 20 to 80% in 13 min while the acetonitrile content was decreased accordingly. In a second step, in which acetonitrile was kept constant at 20%, the concentration of ammonium acetate (pH 4.5) was increased from 0 to 200 mM in 35 min. The eluate was monitored by fluorescence detection (λem, 330 nm; λex, 420 nm). Permethylation of Glycans. The glycan pool (1 nmol) was purified prior to permethylation on a C8 cartridge in order to remove the detergents used for protein deglycosylation (NP-40 and SDS). The cartridge (1 mL) was conditioned with acetonitrile (3 mL) and then water (2 mL). The glycan mixture in solution in water was applied on the cartridge, eluted with 2 mL of water, and dried on Drierite overnight. Permethylation was performed according to the procedure described by Ciucanu and Kerek19 by adding 150 µL of a NaOH/DMSO suspension and stirring the solution at room temperature. After 1 h, 100 µL of iodomethane was added, and the reaction was stirred for another hour. The derivatized oligosaccharides were extracted with chloroform, and (19) Ciucanu, I.; Kerek, F. Carbohydr. Res. 1984, 131, 209-217.
the organic phase was washed with 5% aqueous acetic acid and then four times with water. Electrospray Mass Spectrometry of Permethylated Glycans. The permethylated glycans were dissolved in 100 µL of MeOH/CHCl3 (95:5). This solution was further diluted 1:10 with the electrospray solvent MeOH/H2O (1:1) containing 0.25 mM NaOH. The solution was infused directly into the ion source at a flow rate of 3 µL/min. Profile mass spectra were acquired in the positive ion mode over the mass range of 400-2400 Da in 5.5 s while the cone potential was ramped from 50 to 100 V. Typically 50-70 scans were averaged to yield a mass spectrum. The glycan structures were assigned on the basis of the molecular mass of specific structural motifs. The ES mass spectra were smoothed and integrated using the MassLynx software (Micromass). The peak areas of the multiply charged ions corresponding to one specific component were summed up manually, as the current MaxEnt deconvolution algorithm does not allow processing of complex mixtures containing low-mass components. Most of the signals observed in the ES mass spectra corresponded to sodium adducts and were integrated to generate the glycan profiles. The low-intensity potassium adducts (typically less than 5% relative intensity) were not included in the calculation. MALDI Mass Spectrometry of Permethylated Glycans. Spectra were recorded in the positive ion mode using 20-kV acceleration. The permethylated samples (1 µL, 10 pmol) in solution in MeOH/CHCl3 (95:5) were mixed with 1 µL of matrix solution consisting of R-cyanohydroxycinnamic acid (CHCA) (10 mg) dissolved in 1 mL of MeOH/H2O (1:1) and were applied on a stainless steel target. RESULTS AND DISCUSSION A robust methodology has been designed for characterizing the N-glycosylation pattern of proteins as an alternative to the widely used HPLC approaches. It relies on the mass spectrometric analysis of the permethylated derivatives of N-glycans enzymatically released from proteins. The recombinant soluble CD4 is a 375-amino acid protein, comprising two N-glycosylation sites. In this study, it was deglycosylated with PNGase F and the resulting glycan pool was divided in two fractions for analysis by HPLC on one hand and by electrospray mass spectrometry on the other. The first oligosaccharide fraction was reductively aminated with 2-aminobenzamide, and the fluorescently labeled derivatives were separated by anion exchange HPLC.7 The chromatogram illustrated in Figure 1a shows three major signals between 10 and 35 min corresponding to neutral (B), mono- (BS), and disialylated (BS2) biantennary oligosaccharides, respectively. Two components are detected in the late-eluting peak. The second portion of the glycan pool was permethylated prior to mass spectrometric analysis, using the method reported earlier by Reinhold’s laboratory18 based on the protocol described by Ciucanu and Kerek.19 Derivatization prior to mass spectrometry is mandatory for quantitation as the ionization efficiency of native carbohydrates is poor and depends on the size and the monosaccharide composition of the molecule (e.g., presence of sialic acid). The detergents used for the enzymatic deglycosylation were removed from the glycan mixture by performing a solid-phase extraction with a C8 cartridge. This step is essential to ensure
quantitative permethylation and to reduce background interferences that may considerably affect the quality of the electrospray mass spectra. The derivatized oligosaccharides were extracted with chloroform, and the organic phase was extensively washed with water to remove the salts in order to get reliable and reproducible mass spectra. The permethylated products were analyzed using an electrospray quadrupole mass spectrometer in the presence of a small, well-defined amount of sodium hydroxide (typically 0.25 mM) to ensure exclusive cationization of the oligosaccharides by sodium ions. In this way, one single molecular species was generated for each charge state of a component, which is critical for quantitation as it reduces the complexity of the spectra. The electrospray mass spectrum of the permethylated glycan pool released from the soluble CD4 glycoprotein is shown in Figure 1b. The two distinct envelopes observed reflect the doubly and singly charged sodiated molecular species. The structural assignment of each signal is straightforward as the monosaccharide composition of each component can be easily deduced from its molecular mass. All the signals can be identified at a glance and correspond to biantennary structures (B), with and without fucosylation (BF0-1), with a number of N-acetylneuraminic acid units ranging from zero to two (BS0-2). These results are in agreement with the HPLC analysis but give a much more detailed picture, as the fucosylated and nonfucosylated species are clearly resolved. Similar results were obtained by analyzing the samples by MALDI MS (data not shown). This study aimed at deriving some quantitative information on the abundance of the different components present in the mixture in addition to the identification of the species. Accordingly, the intensities of the signals of the different charge states of one specific component were manually added together as the software used could not perform reliable deconvolution of such complex charge-state distributions. This calculation was performed for all glycoforms, and the data are summarized in Table 1. The values were normalized to the major component, which was set equal to 100%. The monosialylated fucosylated biantennary N-glycan (BFS) constitutes the base peak, and all other species range between 50 and 90%. In a first approximation, it was assumed that all derivatized glycans have an uniform response factor, regardless of their size and monosaccharide composition. Thus, no correction for the response of the different signals was applied. It was also assumed that the response factor does not depend on the charge state. To allow comparison with the HPLC analysis of the 2ABlabeled derivatives, the data were clustered according to the degree of sialylation and then normalized. Both data sets show very good agreement. The reproducibility of the analyses and the validation of the approach by orthogonal techniques are two critical factors for quantitative analysis of complex glycosylation patterns. It encompasses the robustness of the method (i.e., analysis under strictly identical conditions) as well as the ruggedness (i.e., long-term variability due to variation of instrument performance and sample preparation). To assess the reproducibility of the overall methodology, a series of experiments were carried out in triplicate and included all steps, namely, the deglycosylation, the sample cleanup, the derivatization, and the mass spectrometric analyses. The ESMS Analytical Chemistry, Vol. 73, No. 20, October 15, 2001
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Figure 1. HPLC chromatogram of the 2-aminobenzamide-derivatized N-glycans (a) and electrospray mass spectrum of the permethylated oligosaccharides (b) released from recombinant soluble CD4 glycoprotein. Symbols: B, biantennary N-glycan; F, fucosyl residue; S, sialyl residue. 2, sialic acid; solid half-circle, fucose; O, galactose; 9, N-acetylglucosamine; b, mannose. Table 1. Relative Intensities of the Components Identified by Mass Spectrometric and HPLC Analyses of the sCD4 Glycan Pool permethylation and ESMS glycansa
MM
relative abundance
B BF BS BFS BS2 BFS2
2048.3 2222.5 2409.7 2583.9 2771.1 2945.3
52 56 89 100 50 52
normalized intensities 57 100 54
2AB labeling and HPLC tR (min) 14.6 14.8 23.4 23.3 30.2 29.8
normalized intensities 54 100 54
a
Symbols: B, biantennary N-glycan; F, fucosyl residue; S, sialyl residue.
analysis of all three samples was repeated twice. The results summarized in Table 2 illustrate the excellent reproducibility of the method, despite its complexity. Its robustness was established with a variability typically below 10% that accounts for both the sample preparation and the mass spectrometric analysis. Therefore, this approach appears well-suited for the quantitation of the different glycoforms, at least as long as the mixture has limited 4758 Analytical Chemistry, Vol. 73, No. 20, October 15, 2001
Table 2. Reproducibility of the Analytical Procedure (Enzymatic Deglycosylation, Derivatization, ESMS Analysis) Applied to the Glycan Pool of sCD4 Glycoprotein samples glycansa
MM
1
2
3
mean
SD
CV (%)
B BF BS BFS BS2 BFS2
2048.3 2222.5 2409.7 2583.9 2771.1 2945.3
52 58 90 100 45 53
52 53 91 100 46 55
60 58 94 100 45 53
55 56 92 100 45 54
4.6 2.8 2.0 0.0 0.7 1.1
8.4 5.0 2.2 0.0 1.5 2.1
a Symbols: B, biantennary N-glycan; F, fucosyl residue; S, sialyl residue.
heterogeneity and the relative abundance of the components is sufficient to give a distinct signal above the background. Minimal variation over a long period of time is important, particularly if the method is going to be used for monitoring batchto-batch consistency. Thus four samples of the same glycoprotein were analyzed at four different points in time over an 18-month period (Table 3). Here again, for each sample, all preparation steps were repeated and the ESMS analysis was carried out in duplicate.
Table 3. Variability of the ESMS Analyses of the sCD4 Glycan Pool over an Eighteen-Month Period experiments glycansa
MM
1
2
3
4
mean
SD
CV (%)
B BF BS BFS BS2 BFS2
2048.3 2222.5 2409.7 2583.9 2771.1 2945.3
55 56 92 100 46 54
52 56 89 100 50 52
56 54 91 100 46 49
54 56 94 100 44 47
55 55 92 100 45 50
0.8 1.2 1.8 0.0 1.2 3.4
1.5 2.2 2.0 0.0 2.6 6.8
a Symbols: B, biantennary N-glycan; F, fucosyl residue; S, sialyl residue.
Despite the relatively tedious sample preparation, very reproducible results were achieved that demonstrate the ruggedness of the method. The general applicability of the approach was assessed by applying it to other glycoproteins with much more complex glycosylation patterns, such as the human R1-acid glycoprotein (AGP). The HPLC chromatogram of the 2AB-derivatized glycans released from AGP and the electrospray mass spectrum of the permethylated derivatives are shown in Figure 2. Three major
clusters were detected in the 2AB profile with di-, tri-, and tetrasialylated oligosaccharides accounting for 35, 49, and 16%, respectively. This glycan pool is characterized by the presence of highly sialylated tri- and tetraantennary structures. The complexity of each of the three major sialylation clusters reflects the presence of homologous glycans. This is corroborated by the ESMS analysis that showed disialylated biantennary (BS2), trisialylated triantennary (TS3), trisialylated fucosylated triantennary (TFS3), trisialylated tetraantennary (QS3), and tetrasialylated tetraantennary (QS4) glycans as the major components (Figure 2b). At a glance, the mass spectrum allowed the identification of fucosylated structures, which could not be unequivocally resolved by HPLC. The complexity of the spectrum however resides in the presence of several other minor components accounting for the low-intensity signals especially in the high-m/z range. The “manual deconvolution” of this spectrum (i.e., the summation of the intensities of the different charge states corresponding to each component) results in the glycosylation profile shown in Figure 3a. Several minor components including lactosaminyl tetraantennary structures (QLS4, MM 4841.9) were observed. In this case again, a good correlation between the ESMS and the HPLC data was obtained (Table 4).
Figure 2. HPLC chromatogram of the 2-aminobenzamide-derivatized N-glycans (a) and electrospray mass spectrum of the permethylated oligosaccharide derivatives (b) released from human R1-acid glycoprotein. Symbols: X includes biantennary (B), triantennary (T), and tetraantennary (Q) N-glycans; F, fucosyl residue; S, sialyl residue; L, lactosamine unit.
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Figure 3. Glycan distribution derived from the electrospray mass spectra of the permethylated N-glycan pool released from native human R1-acid glycoprotein (a), AGP treated with neuraminidase NANase III (b), and NANase I (c) prior to deglycosylation.
However, it appears that minor components of very heterogeneous samples cannot always be readily identified. To overcome this problem, an alternative, aiming at decreasing the number of components, has been explored. As sialylation represents the major contributor to the sample heterogeneity, the complexity of the mixture can be reduced by treating the glycoprotein with a neuraminidase. Two recombinant neuraminidases, cloned from S. pneumoniae (NANase I) and A. ureafaciens (NANase III), 4760 Analytical Chemistry, Vol. 73, No. 20, October 15, 2001
respectively, were chosen to perform desialylation prior to deglycosylation and permethylation. The glycan pool resulting from the action of the enzyme with the broadest specificity (NANase III) showed a significantly simplified pattern that immediately allows determination of the antennary distribution (Figure 3b). Also, the fucosylated and other minor components, namely, the high-mass glycans, QL, QLF, and QL2, gave rise to very distinct signals and were readily identified.
Figure 4. MALDI mass spectra of the permethylated derivatives of the glycan pool released from human R1-acid glycoprotein treated with neuraminidase NANase III (a) and NANase I (b) prior to deglycosylation. Table 4. Relative Intensities of the Components Identified by Mass Spectrometric and HPLC Analyses of the AGP Glycan Pool permethylation and ESMS glycansa
MM
relative abundance
BS2 TS2 TFS2 QS2 TS3 TFS3 QS3 QFS3 QLS3 QS4 QFS4 QLS4
2771.1 3220.6 3394.7 3673.1 3582.0 3756.1 4031.5 4205.6 4481.0 4392.9 4567.0 4841.9
16 8 3 7 25 9 10 4 2 10 4 2
normalized intensities
2AB labeling and HPLC normalized intensities
Table 5. Sialylation Pattern of Glycan Pool for Native and NANase I- and NANase-III Treated AGP glycansa
native
NANase I
NANase III
X XS XS2 XS3 XS4
0 0 34 50 16
17 48 35 0 0
100 0 0 0 0
% sialylation
87
38
0
68
71
a Symbols: X includes biantennary (B), triantennary (T), and tetraantennary (Q) N-glycans. S, sialyl residue.
100
100
32
33
the relative occurrence of this linkage in the whole pattern (Figure 3c). The data summarized in Table 5 allow to derive quantitative information on the level of sialylation as well as on the sialic acid substitution pattern. The level of sialylation is defined as the percentage of terminal nonreducing galactoses bearing a sialyl residue. It is calculated as the ratio of the sum of the intensities of the sialylated glycans individually multiplied by the corresponding number of carried sialic acid residues to the sum of the intensities of each glycan multiplied by the corresponding number of antennae. Quantitative data derived from the analysis of the partially sialylated glycan pool as the result of NANase I treatment
a N-glycans: B, biantennary; T, triantennary; Q, tetraantennary; F, fucosyl residue; S, sialyl residue; L, lactosamine unit.
The treatment with the enzyme NANase I that removes selectively R(2-6)-linked sialic acid residues yielded a mixture of neutral and partially sialylated glycans giving information about
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Table 6. Antennary Distribution of Glycan Pool for Native and NANase I- and NANase III-Treated AGP glycansa,b
MM
native
NANase I
NANase III
B T TF Q QF QL
2048.3 2497.7 2671.9 2947.2 3121.4 3396.7
48 100 36 81 25 11
51 100 45 83 30 9
42 100 40 76 28 11
a N-Glycans: B, biantennary; T, triantennary; Q, ttraantennary; F, fucosyl residue; L, lactosamine unit. b Only the neutral substructures observed in the analysis of the three different samples are reported.
shows that the relative proportion of NeuAc R(2-6) substitution reaches 38%. By comparison with the degree of sialylation of the native glycoprotein, calculated at 87%, the percentage of the NeuAc R(2-3) linkage is then estimated to be 49%. The same samples were also analyzed by MALDI MS and the spectra yielded the same information (Figure 4). Table 6 reports the antennary distribution for the glycan pool as deduced from the ESMS analyses of the native, partially, and fully desialylated samples. Here again, the good agreement validates the pattern simplification by desialylation for the more complex glycosylation profiles. CONCLUSION The methodology described in this study for profiling glycan pools enzymatically released from glycoproteins relies on permethylation and the subsequent analysis of the oligosaccharide derivatives by mass spectrometry. Although this approach involves a somewhat complex sample preparation, the quality and reproducibility of the data yielded solid qualitative and quantitative information on the glycosylation of the analyzed proteins. First, the molecular mass of the various components allowed determination of the monosaccharide composition and immediate identification of the nature of the various glycoforms. The identification was far more reliable than with HPLC analysis because homologous structures such as fucosylated and nonfucosylated glycans were unambiguously resolved. Both correlation of the data obtained by the ESMS and HPLC methods and reproducibility of the method (typically >90%) were excellent, validating the approach and making it a robust tool for the quantitative, or at least the semiquantitative, assessment of
4762 Analytical Chemistry, Vol. 73, No. 20, October 15, 2001
the glycosylation pattern of proteins. These findings were crucial as they validated the assumption made earlier that, in a first approximation, permethylated glycans have a uniform response factor, regardless of their size and the nature of their constituents. This assumption was justified by the fact that the derivatization used yields to similar chemical compounds. In addition, throughout this study, it was implicitly assumed that the response factor for the different charge states was identical, which also appeared to be true. This might become an issue when dealing with components showing a very broad charge distribution. Moreover, the glycosylation pattern of the proteins was precisely described by the relative abundance of the different components, allowing determination of the antennary distribution and the level of sialylation. However, isobaric components are overlapping and cannot be differentiated by this method. Indeed, high-order structures such as triantennary with one lactosamine unit and tetraantennary glycans are not separated, and ambiguities remain. Finally, in the case of very complex mixtures, it was demonstrated that this strategy can be used in conjunction with exoglycosidases. The profiles obtained after enzymatic desialylation, deglycosylation, and permethylation were simplified, providing a direct readout of the antennary distribution and allowing the identification of minor components, even if the information related to the sialylation was lost. This approach was applied primarily to the characterization of the glycan pool obtained from intact proteins. However, a more precise description of the posttranslational modifications can be obtained by performing a site-specific analysis using the same approach on the glycopeptides generated by a proteolytic digest of the glycoprotein. Thus, the information derived from the analyses and the broad range of application make this methodology a valuable tool for characterizing the glycosylation pattern of recombinant proteins. ACKNOWLEDGMENT The authors acknowledge Werner Meier for the soluble CD4 sample and for helpful discussion.
Received for review July 10, 2001. Accepted August 27, 2001. AC015560A
