Anal. Chem. 2000, 72, 3539-3546
Analysis of Nonderivatized Neutral and Sialylated Oligosaccharides by Electrospray Mass Spectrometry Lihua Huang and Ralph M. Riggin*
Lilly Research Laboratories, Eli Lilly and Company, Lilly Technology Center, Indianapolis, Indiana 46285
An HPLC/MS method has been developed that allows rapid, direct analysis of underivatized sialylated as well as neutral oligosaccharides. The method involves the separation of oligosaccharides from salts and proteins using RP-HPLC with a formic acid/acetonitrile/water mobile phase system and on-line electrospray mass spectrometry analysis in the positive ion mode. Under the solution conditions employed, both neutral and acidic (sialylated) oligosaccharides are protonated and therefore detected. In contrast to MALDI-TOF MS, no loss of sialic acid is observed when operating in the positive ion mode. Furthermore, the capability of this method to provide quantitative estimates of the relative abundance of each oligosaccharide mass has been demonstrated using fetuin as a model compound. Many therapeutic proteins (marketed products as well as those undergoing clinical development) are glycoproteins. The oligosaccharides contained in glycoproteins can have diverse biological roles, and in some cases the oligosaccharide moieties can significantly impact the therapeutic properties of the glycoprotein.1 Therapeutic glycoproteins are usually produced using recombinant DNA cell culture techniques, and since glycosylation is not under direct genetic control, cell culture conditions such as pH, cell density, nutrient concentrations, and metabolite concentrations can affect the distribution of glycoforms present in the glycoprotein product. The glycoform distribution of these products, therefore, must be carefully monitored during product development to ensure that a reproducible product is produced. In addition, any proposed changes to the cell culture process (after the initial product is registered) must be carefully assessed to ensure that the glycoform distribution is not significantly altered. While a variety of techniques are available to assess carbohydrate structure, many of the techniques are either too laborious or time-consuming (e.g. NMR) to be utilized on a repetitive basis or are too nonspecific (e.g. HPLC with fluorescence derivatization) for process development purposes. During the past decade, mass spectrometry (MS) has gained popularity as a technique for assessment of oligosaccharide profiles of therapeutic glycoproteins. While mass spectrometry profiles cannot be used to directly determine anomeric configuration or branching patterns, once these features are established by other means (e.g. NMR, (1) Varki, A. Glycobiology 1993, 3, 97-130. 10.1021/ac0001378 CCC: $19.00 Published on Web 06/20/2000
© 2000 American Chemical Society
methylation GC/MS, etc.) MS data can be used to detect qualitative and, in some cases, quantitative changes in glycoform distribution. Oligosaccharide characterization by MS has been enabled by the development of two ionization methodssmatrixassisted laser desorption ionization (MALDI)2 and electrospray ionization (ESI).3 Each of these ionization modes has certain advantages and limitations as currently applied to glycoprotein characterization. Both ionization modes are frequently used to assess glycosylation profiles by analysis of glycopeptides resulting from trypsin (or other protease) digestion of the protein.4,5 While this approach is highly useful for glycoprotein characterization, there are many situations in which the direct analysis of the oligosaccharides released from glycoproteins (e.g. using PNGaseF) would be desirable. In this circumstance, the applicability of these two ionization modes has been somewhat more limited, requiring a variety of approaches.6 Most of the work on neutral or acidic (sialylated) oligosaccharides has utilized MALDI (either in the positive or negative ionization mode).7 ESI has not been widely used for direct analysis of neutral oligosaccharides due to the observation that under most circumstances ionization is poor; thus most previous work has utilized derivatization prior to ESIMS characterization8 or sodium acetate or ammonium acetate have been added to the solution to enhance ionization.9 Recently, Bahr et al.6 used nanoelectrospray mass spectrometry to characterize underivatized neutral oligosaccharides in the positive ion mode, as sodiated ions. They found that nanoelectrospray ionization greatly increases ESI mass spectral sensitivity, comparable to that of peptides or proteins, for underivatized neutral oligosaccharides, but did not extend their work to detection of sialylated oligosaccharides. (2) Karas, M.; Bachmann, D., Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1987, 78, 53-68. (3) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (4) Medzihradszky, K. F.; Maltby, D. A.; Hall, S. C.; Settineri, C. A.; Burlingame, A. L. J. Am. Soc. Mass Spectrom. 1994, 5, 350-358. (5) Rapp, U.; Resemann, A.; Mayer-Posner, F. J. In Techniques in Glycobiology; Townsend, R. R., Hotchkiss, A. T., Jr., Eds.; Marcel Dekker: New York, 1997; pp 53-65. (6) Bahr, U.; Pfenninger, A.; Karas, M.; Stahl, B. Anal. Chem. 1997, 69, 45304535. (7) Burlingame, A. L.; Boyd, R. K.; Gaskell, S. J. Anal. Chem. 1996, 68, 599R651R. (8) Reinhold, V. N.; Reinhold, B. B.; Costello, C. E. Anal. Chem. 1995, 67, 1772-1784. (9) Duffin, K. L.; Welply, J. K.; Huang, E.; Henion, J. D. Anal. Chem. 1992, 64, 1440-1448.
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For underivatized acidic oligosaccharides, either ESI or MALDI in the negative ion mode has been used.10,11 However, sodium ion adduction and sialic acid loss have been significant problems, particularly for MALDI analysis of acidic oligosaccharides. While addition of spermine has recently been shown to minimize sodium ion adduction,12 loss of sialic acid during the MALDI process remains a significant problem. Furthermore, the requirement to run sialylated oligosaccharides in the negative ion mode and neutral species in the positive ion mode remains a significant limitation in cases where one desires to quantitatively estimate the overall oligosaccharide distribution. In this paper, we describe a rapid and sensitive method to analyze underivatized neutral and acidic oligosaccharides by ESI in the positive ion mode. To our knowledge, this is the first time that ESI-MS has been demonstrated to determine protonated, underivatized, neutral, and acidic (sialylated) oligosaccharide distributions in a single assay. This method appears to offer a simple and perhaps general approach for the rapid assessment of oligosaccharide distribution of glycoproteins. Since both neutral and sialylated oligosaccharides are detected in a single assay, this method offers the potential of rapidly determining overall oligosaccharide distributions, unlike previous approaches in which the neutral oligosaccharides are detected in the positive ion mode and sialylated oligosaccharides are detected in the negative ion mode. Furthermore, the use of ESI rather than MALDI offers a more quantitative means of determining distribution of the various oligosaccharides. The ability of this method to provide quantitative estimates of oligosaccharide distribution has been demonstrated using fetuin as a model glycoprotein. EXPERIMENTAL SECTION Materials. Bovine fetuin from fetal calf serum (lot no. 105H9522) was purchased from Sigma Chemical Co. (St. Louis, MO). Recombinant human activated protein C (rhAPC), expressed in HK-293 cells, was produced by Eli Lilly and Co. N-Glycosidase F was purchased from Boehringer Mannheim (Indianapolis, IN). Other chemicals are analytical grade and commercially available. Methods. Preparation of Free N-Linked Oligosaccharides. A 2 mg amount (in 400 µL of sample solution) of bovine fetuin or rhaPC was mixed with 480 mg of urea, 176 µL of 3 M tris buffer, pH ) 8.0, and 30 µL of 50 mg/mL dithiothreitol (DTT) solution. The mixture was vortexed and incubated at 37 °C for 10-30 min. The reduced glycoproteins were alkylated by mixing 50 µL of 100 mg/mL iodoacetic or iodoactamide solution and incubating at room temperature for 10-30 min. The reduced and alkylated glycoproteins were desalted on a disposable desalting column (Bio-Rad Econo Pak 10DG). The column was washed and equilibrated with about 20 mL of 10 mM ammonium bicarbonate buffer before use. The 2 mL glycoprotein fractions were collected for each sample. N-Glycosidase F solution was added in the ratio of enzyme/glycoprotein to 2-4 units/mg. The solution was incubated at 37 °C for 2-20 h. The reduced and carboxymethylated proteins were precipitated by adding 10% (V/V) acetic acid solution in the ratio of 1/100 (v/v) of acetic acid solution/protein (10) Chai, W.; Luo, J.; Lim, C. K.; Lawson, A. M. Anal. Chem. 1998, 70, 20602066. (11) Papac, D. I.; Jones, A. J. S.; Basa, L. J. In Techniques in Glycobiology; Townsend, R. R., Hotchkiss, A. T., Jr., Eds.; Marcel Dekker: New York, 1997; pp 33-52.
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solution. The supernatant solution was used for mass spectrometry analysis. In the experiment in which both the oligosaccharide and deglycosylated protein mass spectra were recorded, the protein precipitation step was omitted. Desialylation of Fetuin Oligosaccharides. A 1 mL volume of fetuin oligosaccharide solution was dried using a vacuum centrifugation system and then reconstituted in 500 µL of reagent grade water. A 250 µL aliquot of the solution was mixed with 250 µL of 0.2 N trifluoroacetic acid (TFA) solution. The mixture was incubated at 70 °C for 2 h to desialylate the fetuin. The remaining 250 µL of oligosaccharide solution was mixed with 250 µL of reagent grade water, and the mixture was used as the “intact” fetuin oligosaccharide solution (1.0 mg/mL starting fetuin material). The “intact” and “desialylated” fetuin solutions were mixed in proportional volumes to yield solutions having theoretical desialylated fetuin contents of 0, 20, 35, 50, 65, 80, and 100%, respectively. The 70 µL microliter aliquots of these solutions were analyzed by HPLC/MS. Weak Anion Exchange (WAE)-HPLC of Fetuin Oligosaccharides. A 200 µL aliquot of fetuin oligosaccharide solution was dried using a vacuum centrifugation system, and the oligosaccharides were then labeled with 2-aminobenzamide (2-AB) according to the instructions contained in the Oxford Glycoscience Signal 2-AB Glycan Labeling kit (K-404). After labeling, the excess dye was removed using a 1 mL P-2 gel spin column (BioRad Laboratories, Inc.). The resulting solution (500 µl) was analyzed by WAE-HPLC using fluorescence detection. The labeled oligosaccharides were analyzed by WAE-HPLC according to the following conditions. The instrument used for analysis and fraction collection was a Beckman System Gold HPLC system equipped with a model 126 programmable solvent, a model 158 diode array detector, a model 1100 Hewlett Packard autoinjector, a Model F1000 Hitachi fluorescence photometer, and a Oxford Glycoscience GlycoSep C column. The excitation and emission wavelengths of the fluorescence detector were 330 and 420 nm, respectively. The UV detector, which was used to aid fraction collection, was set to A channel ) 350 nm and B channel ) 250 nm. An HP1000 chromatography data system was coupled to the HPLC system to record the fluorescence chromatogram. The HPLC was a gradient system where solvent A was 20% acetonitrile in Milli Q water and solvent B was 250 mM ammonium acetate, pH 4.5, in solvent A. The column was equilibrated with solvent A at 0.4 mL/ min flow rate for ∼20 min before sample injection. After injection (100 µL), the column was eluted with solvent A for 2 min, and then the mobile phase composition was linearly increased to 45% solvent B over 30 min. The mobile phase was then increased to 90% B over 2 min and held for an additional 6 min before decreasing to 0% B in preparation for the subsequent injection. HPLC/MS Analysis. A Beckman System Gold HPLC system equipped with a diode array UV detector and Zorbax SB300 C18, 2.1 × 150 mm, 5 µm particle size column was used for the separation. The column was equilibrated with 0.15% formic acid in HPLC grade water (solvent A) for 10-15 min before injection. Approximately 30-50 µL of the supernatant solution (containing 30-50 µg of starting glycoprotein material) was injected, and the column flow rate was 0.2 mL/min. Following sample injection the (12) Mechref, Y.; Novotny, M. V. J. Am. Soc. Mass Spectrom. 1998, 9, 12931302.
Figure 1. (a) Total ion chromatogram (TIC) of fetuin oligosaccharides and (b) mass spectrum of oligosaccharide peak (region from ∼5-11 min retention time, which includes the 5-9 min elution region in which low-intensity, small oligosaccharides elute).
column was eluted with 100% solvent A for 2 min, and then solvent B (0.12% formic acid in acetonitrile) content was increased from 0 to 15% over 8 min. Following a 4 min hold the mobile phase composition was then rapidly returned to 100% solvent A. In the experiment in which both the released oligosaccharides and the deglycosylated protein were analyzed the elution profile was modified as follows. Following the gradient reaching 15% B the mobile composition was linearly increased to 40% B over 3 min, then to 45% B over 15 min, and finally to 80% B over 2 min. A small stream (about 10 µL/min) of the effluent from the HPLC was diverted to the electrospray mass spectrometer (a PE Sciex model API III). The mass spectrometer was operated using an ion spray voltage of 4800 V, an orifice potential (OR) of 50 V, a step size of 0.33 /step, a scan rate of 6 s/scan, and a scan range of 650-1650 (for fetuin oligosaccharides) and 700-1900 (for aPC oligosaccharides).
Table 1. Data Summary for HPLC/MS of Fetuin Oligosaccharides signals at m/z amu +2H+ +3H+
found mass (Da)
proposed oligosaccharide formula
theoret mass (Da)
967.0 1112.5 1149.8 1295.0 1441.2 1586.7
1932.0 2223.3 2297.6 2588.0 2880.2 3171.2
(NeuAc)1(HexNAc)4(Hex)5 (NeuAc)2(HexNAc)4(Hex)5 (NeuAc)1(HexNAc)5(Hex)6 (NeuAc)2(HexNAc)5(Hex)6 (NeuAc)3(HexNAc)5(Hex)6 (NeuAc)4(HexNAc)5(Hex)6
1932.7 2224.0 2298.1 2589.4 2880.6 3171.9
ND 742.2 ND 863.7 961.0 1058.0
a Abbreviations used throughout all the tables and text are as follows: NeuAc ) N-acetylneuraminic acid; HexNAc ) N-acetyl hexosamine (e.g. N-acetylglucosamine or galactosamine); Hex ) Hexose (e.g. mannose, galactose); Fuc ) fucose.
RESULTS AND DISCUSSIONS The N-linked oligosaccharides of bovine fetuin have been extensively characterized using NMR13 and high-pH anion exchange chromatography with pulsed amperometric detection
(HPAE-PAD).14 The primary components are trisialylated triantennary complex oligosaccharides with R (2f6) or R (2f3) linked sialic acid residues. Tetrasialylated triantennary oligosaccharides and diantennary oligosaccharides are also present. Relative abundances of the oligosaccharides have been reported for various commercially available bovine fetuin materials.13,14 The HPLC/ ESI-MS method was applied to bovine fetuin, and the total ion
(13) Green, E. D.; Adelt, G.; Baenziger, J. U.; Wilson, S.; van Halbeek, H. J. Biol. Chem. 1988, 263, 18253-18268.
(14) Townsend, R. R.; Hardy, M. R.; Cumming, D. A.; Carver, J. P.; Bendiak, B. Anal. Biochem. 1989, 182, 1-8.
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Table 2. Data Summary for HPLC/MS of rhAPC Oligosaccharidesa signals at m/z amu +2H+ +3H+
found mass (Da)
proposed oligosaccharide formula
theoret mass (Da)
(HexNAc)5(Hex)4(Fuc)2 (HexNAc)6(Hex)3(Fuc)2 (NeuAc)3(HexNAc)5(Hex)6(Fuc)1 (NeuAc)1(HexNAc)4(Hex)5(Fuc)1 (SO4)1(HexNAc)6(Hex)3(Fuc)2 (NeuAc)1(HexNAc)5(Hex)4(Fuc)1 (HexNAc)6(Hex)3(Fuc)3 (NeuAc)1(HexNAc)6(Hex)3(Fuc)1 (NeuAc)1(HexNAc)5(Hex)4(Fuc)2 (NeuAc)1(HexNAc)6(Hex)3(Fuc)2 (NeuAc)2(HexNAc)4(Hex)5(Fuc)1 (NeuAc)2(HexNAc)5(Hex)4(Fuc)1 (NeuAc)4(HexNAc)6(Hex)7(Fuc)1 (NeuAc)4(HexNAc)7(Hex)6(Fuc)1
1974.8 2015.9 3026.8 2078.9 2095.9 2120.0 2162.0 2161.0 2266.1 2307.2 2370.2 2411.2 3683.4 3724.5
988.1 1008.6 1514.1 1039.9 1048.5 1061.0 1081.5
ND ND 1009.5 ND ND ND ND
1974.2 2015.2 3025.9 2077.8 2095.0 2120.0 2161.0
1134.0 1154.4 1186.1 1206.6 ND 932.0 (+4H+)
ND ND ND ND 1228.7 1242.5
2266.0 2306.8 2370.2 2411.2 3683.1 3724.3
oligosaccharideb 2a, 2b
1 9 11 7 19 15 24, 25 23
a The charge states of the mass signals were determined on the basis of the difference between the oligosaccharide signal and its water adduct signal. b Named to be consistent with ref 15.
Figure 2. (a) Total ion chromatogram of rhAPC oligosaccharides and (b) mass spectrum of oligosaccharide peak.
chromatogram (TIC) is shown in Figure 1a. The mass spectrum of oligosaccharide signals observed over the range of 5 to 11 min retention time is shown in Figure 1b. On the basis of the published data describing the fetuin N-linked oligosaccharides, the significant mass spectral ions observed can be assigned. All the signals represent protonated molecular ions (predominantly doubly and triply charged) ions are observed. For example, trisialylated triantennary oligosaccharides (NeuAc)3(HexNAc)5(Hex)6, the 3542
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most intense oligosaccharides in bovine fetuin,13 have a molecular weight of 2880.62 Da, and thus, the doubly (+2H+) and triply (+3H+) charged molecular ion signals (m/z) are theoretically 1441.3 and 961.2, respectively, Major signals are found in the mass spectrum (Figure 1b) at m/z 1441.2 and 961.0 (i.e. consistent with the theoretical m/z values). Table 1 lists all the significant signals obtained by HPLC/MS and the corresponding oligosaccharide formula assignments.4,13 Generally the observed masses agreed
Figure 3. Fluorescence WAE-HPLC chromatogram of fluorescence-labeled fetuin oligosaccharides. Table 3. Quantitative Results of Fetuin Oligosaccharides Obtained by HPLC/MS and Weak Anion Exchange (WAE) HPLC of Fluorescence-Labeled (2-AB) Oligosaccharides rel abundance (%) oligosaccharide monosialylated disialylated diantennary disialylated triantennary trisialylated triantennary tetrasialylated triantennary
HPLC/MSa (1.5)b
5.3 21.1 (1.1) 11.7 (0.9) 43.5 (2.3) 18.3 (1.7)
WAE-HPLC 6.3 18.4 9.7 47.6 18.1
a The quantitative results of HPLC/MS were obtained as follows. For each of the primary N-linked oligosaccharides (masses), its theoretical signal at the appropriate m/z position was calculated. A 2.5 m/z range was used to ensure that the entire signal for each oligosaccharide was integrated. The area (cps) of each oligosaccharide is equal to the sum of the areas (cps) of the extracted ion chromatograms of all possible theoretical signals in the scan range. The relative abundance of each oligosaccharide was calculated after obtaining the areas of all oligosaccharides observed. b Mean and (standard deviation) for replicate assays on three separate days.
Figure 4. Mass spectra of fetuin oligosaccharides spiked with known amounts of desialylated fetuin oligosaccharides.
with the theoretical mass within 0.03%. This method was further applied to rhAPC, for which the major N-linked oligosaccharides have been characterized using HPAE/ PAD, derivatization GC/MS, and proton NMR.15 In that study, 12 different oligosaccharide masses (some of which are isobaric structures) were observed, including asialo diantennary to fully sialylated tetraantennary structures. The TIC and corresponding oligosaccharide mass spectrum for rhAPC are shown in Figure (15) Yan, S. B.; Chao, Y. B.; van Halbeek, H. Glycobiology 1993, 3, 597-608.
2a,b, respectively. The mass spectral data and corresponding oligosaccharide structural assignments are summarized in Table 2. All 12 expected oligosaccharide structures are apparent in the spectrum with m/z signals in the range of 900-1200 (representing doubly and triply charged protonated molecular ions). On the basis of these data, the HPLC/ESI-MS method appears to have comparable response for oligosaccharides, regardless of the degree of sialylation. To evaluate whether this apparent observation was valid, the relative abundances of each of the categories of fetuin N-linked oligosaccharides were determined using the HPLC/ESI-MS method as well as a fluorescence HPLC method using 2-aminobenzamide derivatization. The HPLC fluorescence chromatogram is shown in Figure 3, and the apparent relative abundances obtained by both techniques are shown in Table 3. The relative abundances for each oligosaccharide structure obtained by the two techniques agreed very closely, thereby indicating that the HPLC/ESI-MS method has comparable sensitivity for N-linked oligosaccharides irrespective of the degree of sialylation. These values obtained were also in reasonable Analytical Chemistry, Vol. 72, No. 15, August 1, 2000
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Table 4. Distribution (Relative %) of Di- and Triantennary Oligosaccharides in Fetuin Spiked with Desialylated Materiala triantennary
diantennary
quantity of asialo material added
asialo forms
sialylated forms
asialo forms
sialylated forms
0 20 35 50 65 80 100
0 23 39 54 68 78 100
100 77 61 46 32 22 0
0 25 43 55 67 79 100
100 75 57 45 33 21 0
a The relative percent of asialo or sialylated glycoforms was calculated by the following equation: asialo di or tri ) [area of asialo di or tri/(area of asialo di or tri + area of sialylated di or tri)] × 100%, where sialylated diantennary includes mono- and disialylated diantennary oligosaccharides and sialylated triantennary includes di-, tri- and tetra-sialylated triantennary oligosaccharides.
agreement with literature values.13,14 Fetuin obtained from Sigma has been reported to contain diantennary species at 14-30% (compared to 25% in this study) and tetrasialylated triantennary species at 5-14% (compared to 18% in this study). Considerable variation in oligosaccharide distribution was observed between different fetuin lots;14 therefore, comparison of values obtained by two distinctly different techniques on the same fetuin lot (i.e. HPLC/ESI-MS and HPLC/fluorescence derivatization) is a more direct means of assessing relative sensitivity for the various oligosaccharide species. The very good agreement between the two sets of results in Table 3 strongly indicates that the HPLC/ ESI-MS technique offers comparable response for the various oligosaccharides regardless of the degree of sialylation. This finding is presumed to be due to the use of formic acid in the HPLC eluent which ensures that both neutral and acidic oligosaccharides are protonated. While no neutral oligosaccharide structures are present at significant levels in fetuin, neutral oligosaccharides are apparent in the rhAPC material, indicating that the method (as expected) detects both neutral and acidic oligosaccharides in a single analysis. To further study the ability of this method to provide quantitative estimates of neutral and acidic oligosaccharides in a single assay, an experiment was conducted in which fetuin oligosaccharides were mixed in known proportions with completely desialylated fetuin oligosaccharides produced by treatment with dilute TFA. The resulting mass spectra are shown in Figure 4. Signals at m/z 821.3 represents doubly protonated ions of asialo-diantennary oligosaccharide. Signals at m/z 669.5 and 1004.4 represent triply and doubly protonated ions of asialo-triantennary oligosaccharides. Signals at m/z 864.0 and 1295.5 represent triply and doubly protonated ions, respectively, of disialylated triantennary oligosaccharides. Signals at m/z 961.2 and 1441 represent triply and doubly protonated ions, respectively, of a trisialylated triantennary oligosaccharide. The signals at m/z 1112.5 and 742.2 represent doubly and triply protonated ions of a disialylated diantennary oligosaccharide, whereas the signals at m/z 1058.2 and 1586.7 represent triply and doubly protonated ions of a tetrasialylated triantennary oligosaccharide. The individual signals were integrated, and the apparent relative abundances of the various oligosaccharides were calculated. The results obtained, 3544 Analytical Chemistry, Vol. 72, No. 15, August 1, 2000
Table 5. Data Summary Obtained by HPLC/MS for Protein Portion of De-N-glycosylated Fetuin found mass (Da)
proposed oligosaccharide formula
expected fetuin mass Daa
error (%)
38142 38355 38437 38653 38727 38800 39013 39090 39307 39380 39455 39669 39749 39963
(NeuAc)0(Hex)3(HexNAc)3 (NeuAc)2(Hex)2(HexNAc)2 (NeuAc)1(Hex)3(HexNAc)3 (NeuAc)3(Hex)2(HexNAc)2 (NeuAc)2(Hex)3(HexNAc)3 (NeuAc)1(Hex)4(HexNAc)4 (NeuAc)3(Hex)3(HexNAc)3 (NeuAc)2(Hex)4(HexNAc)4 (NeuAc)4(Hex)3(HexNAc)3 (NeuAc)3(Hex)4(HexNAc)4 (NeuAc)2(Hex)5(HexNAc)5 (NeuAc)4(Hex)4(HexNAc)4 (NeuAc)3(Hex)5(HexNAc)5 (NeuAc)5(Hex)4(HexNAc)4
38 137.0 38 354.2 38 428.3 38 645.4 38 719.5 38 793.6 39 010.8 39 084.9 39 302.0 39 376.1 39 450.2 39 667.4 39 741.4 39 958.6
0.013 0.002 0.023 0.020 0.019 0.016 0.006 0.013 0.013 0.010 0.012 0.004 0.019 0.011
a The expected molecular weight of protein backbone of fetuin after reduction, carbamidomethylation, and N-glycosidase F treatment is 37041.0 Da (protein sequence 1-341) (16).
shown in Table 4, are in close agreement with the expected (theoretical) values. Linear regression analysis of the abundance of asialo forms versus spike level was conducted for both the diantennary and triantennary forms. The regression analysis of the diantennary data gave an R2 value of 0.992, a slope of 0.96, and a y-intercept of 0.7% whereas the triantennary data gave an R2 value of 0.996, a slope of 0.98, and a y-intercept of 0.6%. These results clearly demonstrate that this method provides highly accurate estimates of the relative amounts of sialylated and neutral oligosaccharides over a wide range of relative concentrations. The data further strengthen the conclusion that the assay response is relatively independent of the degree of sialylation, in contrast to essentially all previously reported methods. In contrast, all previous studies have used the negative ion mode for detection of sialylated species and the positive ion mode for detection of neutral oligosaccharides. The ability to detect both neutral and sialylated species, with comparable response, in a single assay is a significant advantage as this approach allows the quantitative estimation of overall glycoform distribution. This feature of the method is expected to be particularly important for process development applications since one can readily determine the effect of processing variables on overall glycoform distribution. This method was developed primarily for product quality and process development applications where relatively large amounts of sample are generally available; hence, the sensitivity of the method has not been optimized. In most instances total sample used in the assay was on the order of 1 nmol of glycoprotein. However, preliminary experiments using nanoelectrospray direct infusion as well as capillary HPLC indicate that analysis of glycoproteins at the low picomole level should be possible (data not shown). An additional feature of this method is the ability to characterize both the released oligosaccharides and the deglycosylated protein in a single HPLC-MS run. This feature of the method was demonstrated using fetuin (results shown in Figure 5). After treatment with N-glycosidase F, fetuin still exhibits a complex mass spectrum, due to the fact that O-linked oligosaccharides remain attached to the protein. Fetuin is known to have 4 potential
Figure 5. (a) Total ion chromatogram, (b) mass spectrum of shaded region in panel a, and (c) reconstructed mass spectrum of de-N-glycosylated fetuin solution.
O-linked glycosylation sites, Ser 253, Thr 262, Ser 264, and Ser 323.16,17 Three kinds of O-linked oligosaccharides, trisaccharide (NeuAcGalGalNAc), tetrasaccharide (NeuAc2GalGalNAc), and hexasaccharide (NeuAc2Gal2GlcNAcGalNAc) were found from bovine fetuin.18 On the basis of the expected protein structure16 (1-341, expected mass 37041.0 Da after reduction and carbamidomethylation and deglycosylation by N-glycosidase F) and known O-linked glycoforms, the data obtained were assigned to particular glycoforms as shown in Table 5. The literature (SWISS PROT database) reports that Ser 323 is partially glycosylated and one of the following residues, Ser 253, Thr 262, or Ser 264, is also partially occupied on the basis of the tryptic mapping of bovine fetuin by LC/MS.4 Our data are consistent with these reports according to the assignment of glycoforms. CONCLUSION The method described herein offers significant advantages over existing methods for oligosaccharide characterization. Currently available methods using MALDI or ESMS require neutral oli-
gosaccharides and sialylated oligosaccharides to be detected in two separate runs, using the positive and negative ion modes, respectively. In contrast, the method described in this paper allows both neutral and sialylated oligosaccharides to be detected and their relative abundances to be estimated in a single run. This method is very simple, requiring no sample derivatization prior to analysis and has been demonstrated to give reproducible relative abundances. The data obtained for fetuin indicate that the quantitative estimates of relative abundance for the various oligosaccharides are reliable since they are reasonably consistent with previous literature values and are in good agreement with the results obtained by fluorescence HPLC. Furthermore the quantitative capability of the method has been directly demonstrated by analyzing mixtures having known levels of sialo and asialo fetuin oligosaccharides. While this method is not expected to replace other MALDI or ESMS methods for oligosaccharide characterization in various applications, it does provide a very important means to assess the impact of manufacturing process changes on the relative abundance of oligosaccharides in a rapid,
(16) Dziegielewska, K. M.; Brown, W. M.; Casey, S. J.; Christie, D. L.; Foreman, R. C.; Hill, R. M.; Saunders, N. R. J. Biol. Chem. 1990, 265, 4354-4357.
(17) Carr, S. A.; Huddleston, M. J.; Bean, M. F. Protein Sci. 1993, 2, 183-196. (18) Edge, A. S. B.; Spiro, R. G. J. Biol. Chem. 1987, 262, 16135-16141.
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reproducible, and accurate manner. It should prove to be particularly useful in the areas of product quality assessment and process development, where quantitative accuracy is of considerable importance. ACKNOWLEDGMENT The authors thank Ms. Mary Held, who conducted the free
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oligosaccharide preparation and fluorescence HPLC of fetuin oligosaccharides.
Received for review February 4, 2000. Accepted May 8, 2000. AC0001378
