Anal. Chem. 1997, 69, 2510-2516
Direct Isoform Analysis of High-Mannose-Containing Glycoproteins by On-Line Capillary Electrophoresis Electrospray Mass Spectrometry Bernice Yeung, Thomas J. Porter,*,† and James E. Vath‡
Biopharmaceutical Characterization and Analysis, Genetics Institute, Inc., One Burtt Road, Andover, Massachusetts 01810
A method for the analysis of high-mannose glycoproteins based on capillary electrophoresis and electrospray mass spectrometry (CE-ESI MS) was developed. The combination of UV and MS data allowed for the determination of the identities of glycoform peaks separated by CE, using molecular weight information obtained by ESI MS. The method does not require oligosaccharide release or derivatization, and is applicable for neutral glycans such as high-mannose structures. Two high mannose-containing proteins, ribonuclease B (RNase B) and recombinant human bone morphogenetic protein-2 (rhBMP-2), were used as examples to demonstrate the utility of this technique. Microheterogeneity observed in the CE-UV separation of glycoforms was accounted for by the reconstructed ion chromatograms in ESI MS. Carbohydratespecific reporter ions generated by in-source fragmentation of the intact proteins during ESI was compared to the chromatographic UV results. This analysis may prove to be a useful qualitative or semiquantitative tool for comparing carbohydrate contents among different glycoproteins, among isoforms of a given protein, or in batchto-batch comparisons of biopharmaceuticals. Glycosylation plays an important role in defining the biological and biophysical properties of many proteins. Oligosaccharides are attached to the proteins posttranslationally and differential glycosylation is a major source of protein microheterogeneity.1 The number of glycoforms for each protein is determined by the number of glycosylation sites on the protein, the complexity of glycan structures at those sites, and the degree of occupancy of those sites. The situation can be further complicated when combined with protein heterogeneity, in which the number of glycoforms observed may be influenced by both the carbohydrate and protein heterogeneities. For this study, we define glycoform as any glycoprotein variant that differs in the carbohydrate structure, and isoform as any form of the protein arising from differences in rest of the structure, such as protein sequence. General strategies for glycoform analysis usually involve the primary sequence determination of the deglycosylated protein, in conjunction with structural characterization of the released glycan pool. To obtain further information, glycosylation sites and
glycan structures may be determined from the individual glycopeptides generated from an enzymatic or chemical digest. Recently, several reports on the capillary electrophoretic (CE) analysis of intact glycoproteins have been published,2-4 where basic or borate-containing buffers were utilized for the separation. In this report, CE was used for the separation of isoforms and glycoforms of intact, high-mannose-containing proteins. The analysis was further extended by coupling CE to electrospray mass spectrometry (ESI MS) for on-line molecular weight determination of the separated components. In recent years, CE and CE-MS have emerged as valuable tools for the analyses of biomolecules and small drug molecules, primarily through the use of ESI.5-11 The use of matrix-assisted laser desorption time-of-flight MS (MALDI TOF) as an off-line detection technique for CE has also been reported. In this case, CE fractions can be collected either by a commercial CE instrument directly into vials for off-line MALDI analysis,12 or via coaxial interfaces into vials or MALDI targets.13-16 Hancock et al. reported on the analysis of intact glycoproteins by off-line CE and MALDI TOF, where the molecular weights of some of the glycoforms of ovalbumin and singlechain plasminogen activator were identified.12 Recently, Thibault et al.17 reported on the direct CE-ESI MS analyses of several glycoproteins, in both the intact and digested forms. A dynamic,
E-mail,
[email protected]; fax, (508) 623-2604. Present address: Millennium Pharmaceuticals, 640 Memorial Drive, Cambridge, MA 02139. (1) Dwek, R. A.; Edge, C. J.; Harvey, D. J.; Wormald, M. R.; Parekh, R. B. Annu. Rev. Biochem. 1993, 62, 65-100.
(2) Rudd, P. M.; Scragg, I. G.; Coghill, E.; Dwek, R. A. Glycoconjugate J. 1992, 9, 86-91. (3) Watson, E.; Yao, F. Anal. Biochem. 1993, 210, 389-393. (4) James, D. C.; Freedman, R. B.; Hoare, M.; Jenkins, N. Anal. Biochem. 1994, 222, 315-322. (5) Edmonds, C. G.; Loo, J. A.; Barinaga, C. J.; Udseth, H. R.; Smith, R. D. J. Chromatogr. 1989, 474, 21-37. (6) Vinther, A.; Bjorn, S. E.; Sorensen, H. H.; Soeberg, H. J. Chromatogr. 1990, 516, 175-184. (7) Rush, R. S.; Derby, P. L.; Stickland, T. W.; Rohde, M. F. Anal. Chem. 1993, 65, 1834-1842. (8) Rosnack, K. J.; Stroh, J. G.; Singleton, D. H.; Guarino, B. C.; Andrews, G. C. J. Chromatogr. A. 1994, 675, 219-225. (9) Foret, F.; Thompson, T. J.; Vouros, P.; Karger, B. L.; Gebauer, P.; Bocek, P. Anal. Chem. 1994, 66, 445-4458. (10) Wolf, S. M.; Vouros, P. Anal. Chem. 1995, 67, 891-900. (11) Lu, W.; Poon, G. K.; Carmichael, P. L.; Cole, R. B. Anal. Chem. 1996, 68, 668-674. (12) Chakerl, J.; Hancock, W.; Udiavar, S.; Swedberg, S. Presented at the 44th ASMS Conference on Mass Spectrometry and Allied Topics, Portland, OR, May 12-16, 1996. (13) Weinmann, W.; Parker, C. E.; Deterding, L. J.; Papac, D. I.; Hoyes, J.; Przybylski, M.; Tomer, K. B. J. Chromatogr., A 1994, 680, 353-361. (14) Walker, K. L.; Chiu, R. W.; Monnig, C. A.; Wilkins, C. L. Anal. Chem. 1995, 67, 4197-4204. (15) Foret, F.; Muller, O.; Thorne, J.; Gotzinger, W.; Karger, B. L. J. Chromatogr., A 1995, 716, 157-166. (16) Zhang, H.; Caprioli, R. Presented at the 44th ASMS Conference on Mass Spectrometry and Allied Topics, Portland, OR, May 12-16, 1996.
2510 Analytical Chemistry, Vol. 69, No. 13, July 1, 1997
S0003-2700(96)01117-1 CCC: $14.00
† ‡
© 1997 American Chemical Society
cationic coated capillary and high concentration of formic acid were used in their system for the separation. In this work, we used a linear polyacrylamide-coated capillary in conjunction with an acidic β-alanine buffer for the separation of proteins with N-linked high-mannose structures. The use of such low ionic strength, nonvolatile buffers in low concentrations, along with zero electroosmotic flow, has been reported for CE-ESI MS experiments on proteins.9 These buffers can potentially deliver a greater flexibility in the development of CE-ESI MS methods for highly heterogeneous species, where the performance of neither system is compromised. In this paper, RNase B was first examined as a model protein and then the method was applied to a recombinant glycoprotein, rhBMP-2. EXPERIMENTAL SECTION Materials. rhBMP-2 was produced by Genetics Institute, Inc. Iodoacetamide and dithiothreitol were purchased from Aldrich (Milwaukee, WI); β-alanine and RNase B (bovine pancreatic) were obtained from Sigma (St. Louis, MO); ultrapure glacial acetic acid was from J. T. Baker (Phillipsburg, NJ); sequencing grade formic acid (88%), HPLC grade methanol (MeOH), and acetonitrile (ACN) were purchased from Fisher Scientific (Pittsburgh, PA); HPLC grade trifluoroacetic acid (TFA) was obtained from Pierce (Rockford, IL); and water was distilled and deionized (18 MΩ) using a Milli-Q system from Millipore (Bedford, MA). rhBMP-2 Sample Preparation. The protein was stored at -80 °C in the lyophilized form. A 1 mg sample was desalted by reversed phase HPLC with a Supelco C4 cartridge (Supelguard LC-304, 2 cm; Supelco, Bellafonte, PA), on a Waters 600MS system (Bedford, MA) with a multiphasic ACN gradient where solvents A and B were water and 95% ACN, respectively, with 0.1% TFA in both. The protein peak was collected, frozen in liquid N2, and lyophilized in a SpeedVac vacuum centrifuge (Farmingdale, NY). The dried protein was resuspended in 1.92 mL of buffer containing 0.5 M Tris/6 M guanidine/5 mM EDTA (pH 8.5). An aliquot of 1.2 mL (625 µg) was taken for the reduction reaction, to which a 25-µL volume of 0.1 M dithiothreitol was added. The solution was incubated under argon at 37 °C for 1.5 h and then equilibrated to room temperature for 30 min, and 25 µL of 0.250 M iodoacetamide was then added. The reaction mixture was incubated in the dark at room temperature for 1 h and was then stored at -80 °C until a second desalting step. Aliquots from both the reduced (400 µL, ∼200 µg) and the nonreduced (100 µL, ∼50 µg) samples were desalted, using the same RPLC method as described above. Based on area counts observed at 280 nm, the recoveries for the reduced and alkylated protein was ∼119 µg, and for the non-reduced protein was ∼31 µg. Both samples were dried down in the SpeedVac and were reconstituted with 1 mM acetic acid to concentrations of 5.0 (reduced) and 1.0 mg/ mL (nonreduced). Cation Exchange Chromatography. rhBMP-2 dimer isoforms were fractionated with a NaCl gradient on a MonoS 10/10 column as previously described.18 Capillary Electrophoresis. All CE work was performed using the BioCAP capillaries coated with linear polyacrylamide, with 50µm i.d. in various lengths as stated in each case (BioRad Laboratories, Hercules, CA). The CE buffer was made up of 50 (17) Kelly, J. F.; Locke, S. J.; Ramaley, L.; Thibault, P. J. Chromatogr., A 1996, 720, 409-427. (18) Rathore, S.; Hammerstone, K. M.; Dansereau, S.; Porter, T. J. Protein Sci. 1995, 4, 140.
mM β-alanine adjusted to pH 3.5 using acetic acid. All off-line work was carried out using a BioFocus 2000 CE system, equipped with a 50-cm length of capillary installed in a user-assembled capillary cartridge (BioRad Laboratories). Samples were pressure injected for various lengths of time (see figure captions), and a running voltage of 18 kV (360 V/cm) was used. The capillary and the sample compartment were thermostated at 20 °C. For on-line coupling to MS, a home-built CE system consisted of a Spellman CZE 1000R HV supply (Spellman High Voltage, Plainview, NY) was utilized. The lengths of capillaries used were between 65 and 68 cm. The HV electrode was a 1.0-mm-diameter platinum wire (Fisher Scientific) inserted through a septum into the electrolyte vial. For on-line work, samples were injected by siphoning at +15 cm of height relative to the electrolyte (anode) vial. For the 50-µm-i.d. capillaries, this height produced a linear velocity of ∼0.5 nL/s, based on which injection amounts were calculated. The effective CE voltage applied varied between +20.5 and +24.5 kV (see figure captions), producing a field of 315-360 V/cm. All on-line separations were carried at ambient temperature. MS and CE-MS. A VG Platform single quadrupole instrument equipped with a VG ESI source (Micromass, Manchester, U.K.) was used for all work. Calibration was performed using a sodium iodide solution (1 mg/mL) at 1 unit mass resolution. Flow injection work was carried out using the regular ESI probe, whereas CE-MS was set up using a coaxial CE-MS interface supplied by Micromass. The liquid sheath was made up of 75/ 25/0.1 (v/v/v) MeOH/water/formic acid, delivered to the probe tip at 3 µL/min by a syringe pump (Model 22, Harvard Apparatus, South Natick, MA). High-purity N2 gas was supplied at ∼20 L/h as the sheath gas, and no bath (curtain) gas was used. The anode reservoir (injection end) was adjusted to 2 cm higher than the cathode (ESI end), in order to compensate for the pressure differential between the two ends of the system. A schematic of a similar experimental set-up can be found in the literature.19 An ESI voltage of +4.5 kV was applied to the capillary tip, and the cone voltage was set at 30 V for full-scan analyses and 200 V for single-ion monitoring of the reporter ions. Source temperature was set to 80 °C for effective desolvation of the sprayed liquid. Full-scan analyses were carried out at a rate of 500 amu/s for RNase B and 1000 amu/s for rhBMP-2. RESULTS AND DISCUSSION Model Protein: RNase B. As RNase B possesses an isoelectric point and glycan structures similar to rhBMP-2, development of the CE and CE-MS methods was carried out using this protein. Typical CE-UV and CE-MS profiles are given in Figure 1, which show great similarity to each other. The relatively good resolution of the glycoform peaks allowed adequate full-scan analysis by MS to be performed on each peak at 500 amu/s. Summed mass spectra from the labeled peaks in the total ion chromatogram (TIC), as shown in Figure 2, indicate the detection of all the oligomannose structures. Despite some degree of in-source fragmentation at the optimized cone voltage of 30 V, which led to multiple peaks in some mass spectra resembling the previously eluting glycoforms, the MS results unequivocally identified each of the five glycoforms with high accuracy, as indicated in Table 1. (19) Thompson, T. J.; Foret, F.; Vouros, P.; Karger, B. L. Anal. Chem. 1993, 65, 900-906.
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Figure 2. Summed mass spectra from the labeled peaks in the TIC in Figure 1b, taken at a cone voltage of 30 V. In each spectrum, the prefix A refers to the multiply charged ions that represent the molecular mass of the glycoform detected in each instance. Table 1. CE-MS Results of RNase B mass of protein Figure 1. (a) CE-UV profile of RNase B glycoforms. Protein concentration was 1 mg/mL; injection was for 10 psis, which resulted in an amount of ∼0.9 pmol. (b) TIC of a CE-ESI MS analysis of RNase B. Protein concentration was 10 mg/mL, and injection was done for 15 s at 15 cm of height. Approximately 5pmol of protein was injected. Capillary length was 68 cm, and the effective CE voltage was +24.5 kV.
Recombinant Protein: rhBMP-2. rhBMP-2 is a covalently linked dimeric protein that has been found to induce bone growth in vivo in several animal models.20 The sequence of the monomer is shown in Figure 3. The unpaired cysteine (Cys) at position 78 is structurally analogous to that of the Cys that forms the intermolecular disulfide bond in TGF-β2, a cytokine that has seven Cys residues which align with those of rhBMP-2.21 The mature form of rhBMP-2 has a glutamine (Gln) residue at the N-terminus. Cyclization of this Gln residue to pyroglutamic acid (pyroGlu) yields an isoform designated as the pyroGlu form. Additionally, the recombinant-derived protein may also contain another 17amino acid sequence starting at the N-terminal threonine (Figure 3). Therefore, a total of six dimer isoforms may be found in this recombinant protein, with each of them carrying glycoforms derived from the high-mannose glycans. Each of the six dimers is active in vitro and in vivo.18 Previously, the purified mature form of the rhBMP-2 dimer was analyzed by CE using an acidic phosphate buffer, which shows the separation of nine glycoforms that are speculated to be the dimeric combinations of different glycoform structures.22 Using conditions developed with RNase B, CE experiments were next carried out on rhBMP-2. The CE profile of the intact (20) Wozney, J. M.; Rosen, V.; Celeste, A. J.; Mitsock, L. M.; Whitter, M. J.; Kriz, R. W.; Hewick, R. M.; Wang, E. A. Science 1988, 242, 1528-1534. (21) Schlunegger, M. P.; Grutter, M. G. Nature 1992, 358, 430-434.
2512 Analytical Chemistry, Vol. 69, No. 13, July 1, 1997
carbohydrate structure
calculated average
observed
% SD
(HexNAc)2Hex5 (HexNAc)2Hex6 (HexNAc)2Hex7 (HexNAc)2Hex8 (HexNAc)2Hex9
14 899 15 061 15 223 15 385 15 547
14 899 15 061 15 226 15 392 15 554
0.00 0.00 0.019 0.045 0.045
homodimer is given in Figure 4a, which shows very poor resolution for the three isoforms and their glycoforms. CE-MS experiments on the homodimer showed poor sensitivity, and detection in the high m/z range (m/z 2500) was required (data not shown). Taken collectively, these data suggested a tight conformation for the homodimeric protein, which inhibited the addition of charges to the protein molecule during CE and ESI. After reduction and alkylation, a much improved separation was obtained for rhBMP-2 (Figure 4b). Not only were the three N-terminal isoforms now clearly separated but the individual glycoforms within each isoform were also well resolved. Based on migration pattern and relative abundance, the three isoforms were deduced to be the extended form (I), the mature form (II) and the pyroGlu form (III) of the protein. The CE-MS analysis of the reduced rhBMP-2 resulted in a TIC with a profile similar to the UV electropherogram (Figure 5). The summed mass spectra of peaks I-IV are shown in Figure 6. As indicated by the masses detected, peaks I-III were identified to be the extended, mature, and pyroGlu forms of rhBMP-2, respectively. An additional peak, labeled as peak IV, was also observed in the TIC, and the identity of this peak will be discussed below. The CE-MS results from the mass spectra of peaks I-III are summarized in Table 2. (22) Yim, K.; Abrams, J.; Hsu, A. J. Chromatogr., A 1995, 716, 401-412.
Figure 4. CE-UV analysis of (a) rhBMP-2 dimer, with protein concentration of 1.0 mg/mL and an injection for 10 psi s (∼0.47 pmol injected). (b) rhBMP-2 monomer, obtained after reduction, alkylation, and desalting. Protein concentration was 5.0 mg/mL, and the injection was for 2 psi s (∼0.94 pmol injected).
Figure 3. Primary structure and predicted disulfides of rhBMP-2 monomer, based on the homology to TGF-β2.21 The thicker lines represent the disulfide bonds. The thinner lines are added for clarity and denote connectivity between two adjacent amino acids. The darker amino acid residues represent the additional sequence for the extended form. The symbols at N56 represent the high-mannose glycan structure.
The summed mass spectrum under peak IV (Figure 6d, IV) shows masses that correspond to both the mature and pyroGlu forms of the protein. A comparison of electropherograms taken on different days of the reduced protein after initial sample preparation is displayed in Figure 7. From these data, it would appear that peak IV was increasing in abundance relative to the three isoform peaks, and that, after a 5-fold dilution of the sample, the relative intensity of peak IV diminished. Based on the observations in CE-UV and CE-MS, and the fact that the protein had been alkylated with iodoacetamide, it was speculated that peak IV was a noncovalent aggregate of either or both the mature and the pyroGlu forms, and its formation was both concentration and time dependent. During the ESI process, this aggregate would break apart, and therefore, only the monomeric masses were detected. Next, the 5-fold diluted sample was analyzed by CEMS (Figure 8), where peak IV was no longer detected. The mass spectra from this analysis are shown in Figure 9, and masses similar to those in Figures 6, I-III, and Table 2 were identified. The results from Figures 8 and 9 clearly show that only the noncovalent aggregate, peak IV, was missing upon sample dilution, while the three isoforms of the protein remained. From these observations, it appears that formation of the aggregate for rhBMP-2 may have been a result of the high sample concentration required for CE-MS analysis. In practice, large injection volumes are generally undesirable in CE as they result in longer starting
Figure 5. TIC of a CE-MS analysis of the monomeric rhBMP-2, where injection was carried out for 30 s at 15 cm height. Injection amount was estimated to be 5.0 pmol. Capillary length was 65 cm, and the effective CE voltage was +20.5 kV.
zones which in turn lead to poorer resolution.23 In addition, the sensitivity mismatch between CE and MS also demands the use of high sample concentrations. Therefore, the concentrations for both RNase B and rhBMP-2 in CE-MS experiments were relatively high (300-600 µM), which was necessary to ensure that sufficient sample amounts would be injected for adequate detectability in MS. However, as no evidence of aggregation was seen for RNase B at the high concentration, it may be speculated that the reduced and alkylated rhBMP-2 was more prone to aggregation under these buffer conditions. Although a 5-fold diluted sample of rhBMP-2 no longer showed the presence of the aggregate in the TIC, the signal-to-noise ratio for this analysis suffered slightly due to a smaller sample load than in Figure 5. (23) Kila`r, F.; Hjerte´n, S. J. Chromatogr. 1989, 480, 351-357.
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Figure 6. Summed mass spectra from the labeled peaks in the TIC in Figure 5. Table 2. CE-MS Results of rhBMP-2 Monomers carbohydrate structure (HexNAc)2Hex5 (HexNAc)2Hex6 (HexNAc)2Hex7 (HexNAc)2Hex8 (HexNAc)2Hex9 (HexNAc)2Hex5 (HexNAc)2Hex6 (HexNAc)2Hex7 (HexNAc)2Hex8 (HexNAc)2Hex9 (HexNAc)2Hex5 (HexNAc)2Hex6 (HexNAc)2Hex7 (HexNAc)2Hex8 (HexNAc)2Hex9
mass of protein calculated average observed Extended Form (Peak I) 16 503 16 506 16 665 16 668 16 827 16 826 16 989 16 987 17 152 17 155 Mature Form (Peak II) 14 521 14 683 14 845 15 007 15 170
% SD 0.018 0.018 -0.0059 -0.012 0.017
14 516 14 684 14 853 15 007 15 176
-0.034 0.0068 0.054 0.00 0.040
PyroGlu Form (Peak III) 14 504 14 509 14 666 14 668 14 828 14 835 14 990 15 000 15 153 15 152
0.034 0.014 0.047 0.067 -0.0066
Nevertheless, data quality was maintained in the summed mass spectra as shown in Figure 9. The use of transient capillary isotachophoresis12 for on-line concentration of a large injection volume may help to alleviate the problem described. Finally, replacement of the quadrupole MS with a TOF detector should provide a further solution to the general problems associated with the detectability of CE-separated peaks, as the latter has higher data sampling efficiency. 2514 Analytical Chemistry, Vol. 69, No. 13, July 1, 1997
Figure 7. Comparison of the CE-UV profiles of monomeric rhBMP2, showing dependencies of the protein aggregate (peak IV) on both sample concentration and time: (a) 5.0 mg/mL, 1 day after sample preparation, 2 psi s injected; (b) 5.0 mg/mL, 19 days after sample preparation, 2 psi s injected; (c) 1.0 mg/mL, 21 days after sample preparation, 10 psi s injected; (d) 0.2 mg/mL, 21 days after sample preparation, 10 psi s injected.
Figure 8. CE-MS analysis of a diluted sample of the reduced and alkylated rhBMP-2. Protein concentration was 1.0 mg/mL, and injection was performed for 60s at 15 cm height, which introduced ∼2 pmol of sample. The capillary used was 68 cm long, and the effective CE voltage applied was +24.5 kV.
Since the glycoform peaks of rhBMP-2 were not as resolved as those of RNase B in CE-UV, and since a large injection amount was necessary for CE-MS, the microheterogeneity observed in Figure 4b was no longer achievable in the TIC despite a faster scan rate (1000 amu/s). Nevertheless, the separation of glyco-
Figure 11. Examples of the carbohydrate-specific reporter ion SIM results from CE-MS analyses of (a) RNase B (10 mg/mL) and (b) rhBMP-2, 1.0 mg/mL. Both injections were for 45 s at 15 cm height. The sums of the Hex+ ions (upper traces) and HexNAc+ ions (lower traces) are shown.
Figure 9. Summed mass spectra from the labeled peaks in Figure 8.
Figure 10. Total and reconstructed ion chromatograms for the extended form of rhBMP-2 (peak I), taken from the data in Figure 6a.
forms can still be seen in the reconstructed ion chromatograms in Figure 10, where the most intense ion of each glycoform series of the extended form was plotted against the TIC. Again, the use of a nonscanning detector, such as a TOF MS, may help to provide a clearer TIC profile that shows the microheterogeneity from the CE separation.
Carbohydrate-Specific Reporter Ions. Generation of carbohydrate-specific reporter ions was previously demonstrated by Carr et al.24 by collision-induced dissociation and in-source fragmentation in glycopeptide mapping by LC-MS. For in-source fragmentation, it has been shown that sugar oxonium fragment ions may be obtained in the low m/z range and monitored during the LC-MS24 or CE-MS17 peptide map by ramping the sample cone voltage, thus identifying the glycopeptides in the map by full-scan or single-ion monitoring (SIM) using a single-stage MS alone. Similarly, in-source fragmentation may be performed on intact glycoproteins by using a high sample cone voltage.25 This voltage was determined by flow injection analysis of RNase B, where the intensities of the oxonium ions of hexose (Hex+, m/z 163) and N-acetylhexosamine (HexNAc+, m/z 204), as well as their related ions from losses of water, were compared. In the end, a cone voltage of 200 V was found to yield the most abundance of these ions at a source temperature of 80 °C. This cone voltage was the maximum value allowed on the Platform instrument. Based on experience by others,25 better signal-to-noise ratios may be achieved at a lower cone voltage by increasing the source temperature. However, since CE would be the method of sample introduction in this work, source temperatures higher than 80 °C were avoided out of concern for the stability of the capillary coating. The in-source fragmentation of RNase B by CE-MS was carried out using SIM of the reporter ions described earlier. The SIM signals of Hex+ (m/z 163) and two ions from losses of water (m/z 145 and 127) were summed and plotted in the upper trace in Figure 11a, and similarly for the sum of the HexNAc+ ions (m/z 204, 186 and 168) in the lower trace. The similarity of these traces indicates the specificity of detection based on these reporter ions. Three injections of different amounts were carried out for RNase B, where the total Hex+/HexNAc+ area ratio taken from the combination of all five glycoform peaks was found to be consistent (0.8349, SD ) 1.7%, n ) 3). The same experiment was also performed for rhBMP-2. Three injections for different lengths of time were carried out on the 1.0 mg/mL sample (Figure 11b), and the Hex+/HexNAc+ area ratio taken from all three isoforms as a whole was also found to (24) Huddleston, M. J.; Bean, M. F.; Carr, S. A. Anal. Chem. 1993, 65, 877884. (25) Mazsaroff, I.; Yu, W.; Kelley, B.; Vath, J. Anal. Chem. 1997, 69, 25172524.
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Table 3. Area Counts from A280 and SIM Analysis rhBMP-2 isoform extended form mature + pyroGlu forms
% area from A280 % area from SIM (n ) 3) 29 71
40 ( 2.1 60 ( 2.1
be consistent among the different runs (0.7528, SD ) 0.91%, n ) 3). As the number of HexNAc residues are constant for both highmannose proteins, comparison of the Hex+/HexNAc+ area ratios between RNase B and rhBMP-2 indicates a lower amount of mannose residues for the latter, which in turn hints at a more processed oligomannose structure for rhBMP-2 relative to RNase B. In addition to comparison between different glycoproteins, comments may also be made about the degree of glycosylation among isoforms within the same protein, using these carbohydratespecific reporter ion ratios. To further examine this possibility, the same sample of rhBMP-2 was analyzed by cation exchange chromatography, where area counts obtained at λ ) 280nm (A280) showed the percent composition of the three isoforms in the sample independent of their carbohydrate contents (Table 3). Next, the Hex+ and HexNAc+ area counts from SIM were each normalized against the A280 results in order to correct for variations in injected amounts.25 Using the normalized SIM area counts, the final Hex+/HexNAc+ ratio were also found to be consistent (0.5945 ( 2.0% for the extended form and 0.8776 ( 1.02% for the mature and pyroGlu forms combined). On the basis of these results, one can confidently and directly compare the area counts obtained from A280 and SIM, as shown in Table 3, and qualitatively deduce that the extended form of rhBMP-2 contains a higher carbohydrate content than either the mature or pyroGlu form. These results suggest that the presence of the 17-amino acid extension on the extended form influences the degree of processing for the oligomannose species. Alternatively, it was also possible that the differential glycosylation observed could be the result of speciation during purification of the rhBMP-2 glycoforms. CONCLUSIONS In this paper, an analysis of high-mannose-containing glycoproteins by CE-ESI MS is presented. By combining UV and MS data, information on the structures of CE-separated isoform and glycoform peaks can be obtained, even if the glycans are of neutral charges. The results show that isoforms of glycoproteins may be separated and analyzed without the need for oligosaccharide release, derivatization, or labeling. Although the sensitivity mismatch in UV and MS detections may prevent the microheterogeneity to be directly observed, reconstructed ion chromato-
2516 Analytical Chemistry, Vol. 69, No. 13, July 1, 1997
grams show the details in the separation and migration patterns of each glycoform, thus allowing one to comment on observations in the CE-UV profile. Concentration- and time-dependent protein aggregation in a concentrated protein sample has been observed and identified on the basis of results from both CE-UV and CE-MS. As reported here, the presence of the aggregate for rhBMP-2 may be minimized by dilution of the sample by 5-25-fold. Examination of the 5-fold diluted protein sample by CE-MS was still feasible, and it showed the definite absence of the aggregate. This result shows the potential of using CE and CE-MS to monitor protein aggregation, but may also present a limitation for CE-MS in terms of the concentration and buffer requirements, as such dependencies by an aggregate may vary for different proteins. Reporter ions for carbohydrates may be generated by in-source fragmentation and detected in the SIM mode for the CE-separated isoforms. This work represents one of the first examples25 of using reporter ions generated from intact glycoproteins in ESIMS and is certainly the first such application by CE-MS. These carbohydrate-specific reporter ions can be utilized in two ways. First, an internal ratio, such as Hex+/HexNAc+, can be compared among different proteins to indicate the size and complexity of their N-linked glycans. Second, this ratio can be normalized to a protein-only signal (such as A280) for comparisons of carbohydrate contents among isoforms. Examples of such utilities are presented in this paper for rhBMP-2, and the results indicate a higher carbohydrate content for the extended form than the other two isoforms. Similar experiments were performed for a complex carbohydrate-containing glycoprotein where the SIM signal for sialic acid was monitored.25 This approach in combination with CE-MS may prove to be a useful qualitative and semiquantitative tool for comparing carbohydrate contents among glycoproteins and their isoforms, and for detailed comparisons for biopharmaceuticals. ACKNOWLEDGMENT We thank Yuriy Dunayevskiy, Diane Rindgen, and Toni Thompson for valuable discussion on CE-MS operations. Kalvin Yim is acknowledged for the initial concept of this work. We also thank Suman Rathore and Guarev Gupta for technical help on rhBMP-2, and Istvan Mazsaroff for discussion on the data related to reporter ions. Received for review November 1, 1996. Accepted April 4, 1997.X AC9611172 X
Abstract published in Advance ACS Abstracts, May 15, 1997.
