Mass Spectrometry-Based Glycoproteomic Approach Involving Lysine Derivatization for Structural Characterization of Recombinant Human Erythropoietin Mario Cindric´ ,*,† Laura Bindila,‡ Tina C ˇ epo,*,† and Jasna Peter-Katalinic´ ‡ PlivasResearch & Development Ltd., Prilaz baruna Filipovic´a 29, 10000 Zagreb, Croatia, and Institute for Medical Physics and Biophysics, Robert Koch Strasse 31, University of Mu ¨ nster, 48149 Mu ¨ nster, Germany Received April 19, 2006
Lysine-containing peptides comprising glycosylation sites derived from recombinant human erythropoietin (rHuEPO) by trypsin or Lys-C and PNGase F dual digestion were derivatized with 2-methoxy4,5-dihydro-1H-imidazole and its deuterated analogues. In the same reaction, under reducing conditions (β-mercaptoethanol), cysteines were converted into methyl-cysteines and lysines into Lys-4,5-dihydro1H-imidazole. Both modifications on cysteines and lysines simplified the CID-MS/MS spectra, while preserving the structural information by yielding y-series ions and improved the mass spectral signal intensity up to 25 times. Moreover, by this approach, the N-glycan occupation sites were unambiguously determined. O-Glycosylation sites as well as O-glycan structures were determined by a LC-MS/MS experiment carried out on dually digested rHuEPO. N-Glycan mixture purified on a graphitized carbon column using a newly developed method that extracted only sialylated carbohydrates was analyzed first using MALDI-TOF in negative linear ion mode with low mass accuracy but without interferences and metastabile ions and then areflectron with high mass accuracy. After defining the precursor ions, we performed the nanoESI QTOF MS/MS analysis on N-glycans, mainly targeting the distinction between carbohydrates with sialylated antennae and those lacking sialic acid moieties. Keywords: rHuEPO glycosylation • Lys-4,5-dihydro-1H-imidazole • N-glycans • O-glycopeptides • MALDI-TOF • LCMS/MS • nanoESI MS/MS
Introduction The analysis of post-translational modifications is one of the main goals of modern protein structure/activity research.1-5 To understand glycoprotein function and the role of glycosylation in highly glycosylated proteins, which contain N- and O-glycans, the fundamental prerequisite is the structural elucidation of both protein and its glycan moieties. To achieve this, analytical methods capable to provide high sensitivity determination are required. A large palette of mass spectrometry (MS)-based approaches were reported as viable for investigation of glycosylation in glycoproteins.6,7 Satomi et al.8 characterized commercial human transferrin by means of MALDI- and ESI MS/MS analysis of glycopeptides derived by lysylendopeptidase treatment. The method allowed 76% protein sequence coverage and the identification of already known and of a new N-glycosylation site,8 while by LC-ESIMS and MS/ MS site-specific profiling the identity and structure of N-glycans could be fully deduced.9 An elegant method was recently * To whom correspondence should be addressed. M. Cindric´, Plivas Research & Development, Ltd., Prilaz baruna Filipovic´a 29, 10000 Zagreb, Croatia. Phone: +38513723747. Fax: +38513721514. E-mails: mario.cindric@ pliva.hr (for M.C.) or
[email protected] (for T.C.). † PlivasResearch & Development Ltd. ‡ University of Mu ¨ nster.
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reported by Neususs et al. for recombinant human erythropoietin (rHuEPO) intact glycoform profiling. The on-line CE/ ESI TOF MS permits the detection of separated intact glycoform ions and their assignment based on molecular mass calculation. In a single and fast experiment, the identity of a high number of glycoforms can be derived.10 However, for detailed analysis of glycoproteins, the profiling of intact glycoforms assumes already known protein sequence and glycosylation sites. Even in the context of high mass accuracy, the inherent diversity of possible glycan structures within the glycoforms still requires fragmentation studies as indispensable to define the structural architecture, that is, branching, glycan linkage, Neu5AcR2-3 versus Neu5AcR2-6 linkage, presence of disialo element, and so forth. In the present study, we aim the designing of a MS-based approach for detailed structural investigation of rHuEPO glycoprotein: protein sequence with high coverage, N- and O-glycan variants, and their attachment sites within the protein. An analytical step, 2-methoxy-4,5-dihydro-1H-imidazole derivatization of the lysine-containing peptides resulted from protease treatment, was introduced.11 This step increased the accessibility of these peptides to MS and MS/MS investigation leading to sequence coverage higher than 95%, and concurrently allowed identification of N-glycosylation sites. The derivatiza10.1021/pr060177d CCC: $33.50
2006 American Chemical Society
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Structural Characterization of rHuEPO
tion preserves the O-glycans on the peptides; thus, extensive information of O-glycosylation site and O-glycan structures can be disclosed by fragmentation. Mapping and sequencing of the N-glycan mixture resulted after N-deglycosylation further completed the structural data on rHuEPO glycoprotein. The distinct attributes of this method over the existing ones reside primarily in the possibility to gather a higher number of structural data, thus, rendering a deeper insight into glycoprotein structure. This is particularly advantageous for the analysis of unknown glycoproteins. Our option for different MS techniques, MALDI TOF, ESI-QTOF, Q-TRAP, and LC-MS/MS, was guided by the basic requirements for a detailed structural elucidation of glycoproteins: sensitivity, accuracy and resolution, reliable heterogeneity assay, and efficient MS2 and MSn fragmentation. Recombinant human erythropoietin (rHuEPO) produced in Chinese Hamster Ovary (CHO) cells is a highly glycosylated macromolecule containing bi-, tri-, and tetraantennary N-glycoforms (Asn24, Asn38, and Asn83) carrying one to four terminal sialic acid moieties, and sialylated O-glycans as well.12,13 Protein sequence coverage after trypsin or Lys-C digests of highly glycosylated proteins frequently exhibits low values in comparison to coverage after the additional PNGase F Ndeglycosylation.3 Deglycosylation also provides further structural analysis of N-glycosylation sites by MS/MS or MS3 of lysine-containing peptides tagged with 2-methoxy-4,5-dihydro1H-imidazole preferring y-series ions formation of high intensity.11,14,15 Such a direct approach to N-glycans analysis that includes only few steps, namely, PNGase F N-deglycosylation, carbohydrate desalting, and purification, followed by mass spectrometry analysis, was not in the focus of previous rHuEPO analytical protocols.16-18 A graphitized carbon column was previously used as a chromatographic column for rHuEPO N-glycans preceding the on-line LC-MS analysis after the carbohydrate reduction.16,18,19 Other chromatographic techniques compatible with mass spectrometry or NMR are anionexchange and NH2-bonded stationary phase chromatography. Both techniques require additional desalting steps and higher amounts of starting material, while derivatization of carbohydrates may lead to unwanted structural changes and to sample loss.20,21 Therefore, underivatized carbohydrates prepared by a minimum of purification steps and directly analyzed by MALDI-TOF and nanoESI QTOF MS and MS/MS in the negative ion mode at the low picomole level in conjunction with NMR data12,13 can provide all basic structural data, such as number, type, antennarity, and branching patterns of the rHuEPO-derived N-glycans. In contrast, due to the lack of an universal O-glycosidase, O-glycosylation status can be studied on already N-deglycosylated proteins following the tryptic digestion.22,23 In this case, the on-line LC-MS/MS analysis of a trypsin-digested glycoprotein allows selection of glycopeptides containing O-glycans as precursor ions and subsequent fragmentation. By defining the diagnostic ions with precisely assigned fragments, we could determine the differences between two Ser126-linked Oglycans: a trisaccharide Neu5AcR2-3Galβ1-3GalNAc and a tetrasaccharide Neu5AcR2-3Galβ1-3(Neu5AcR2-6)GalNAc. With this strategy, all rHuEPO N- and O-glycans could be straightforwardly identified by MS and MS/MS. This sensitive analytical method depicts a general strategy feasible for analysis of highly glycosylated proteins.
Methods and Materials Preparation of N-Glycans. Recombinant PNGase F, glycerol free, (Roche, Mannheim, Germany) was used in the sample preparation procedure. Samples of rHuEPO purchased from European Pharmacopoeia (Strasbourg, France) were diluted in 20 mM sodium bicarbonate (pH 8.2) to a volume of 10 µL at a concentration of 1 mg/mL. A volume of 0.4 µL of PNGase F recombinant, glycerol free (1 U/mL), was added to samples, and the resulting solutions were incubated at 37 °C for 16 h. After the enzymatic deglycosylation process, protein was precipitated with 100 µL of ethanol at -20 °C for 8 h and separated by centrifugation at 2300g for 30 min. The supernatant containing sugars was evaporated in a SpeedVac at 40 °C for approximately 4 h and resuspended in 10 µL of water prior to purification on a graphitized carbon column. Preparation of Desialylated N-Glycans. Dried N-glycans were resuspended in 10 µL of water and 4 µL of 250 mM sodium phosphate buffer that was additionally added (pH 6). A volume of 2 µL of NANase II (Bio-Rad, Hercules, CA) was mixed with the solution and incubated at 37 °C for 1 h. After desialylation, carbohydrates were purified on a graphitized carbon column. Graphitized Carbon Column Purification. Carbon column material (Charcoal) was collected from commercial kits made by Harvard Apparatus (Holliston, MA). Approximately 3 mg of dry carbon column material was suspended in 200-300 µL of 80% acetonitrile in 0.1% TFA (v/v).7,24 A 200 µL plastic tip was filled at the bottom with a small amount of filter paper (glass microfiber filter, Whatman, Maidstone, England). The tip was additionally filled with 50 µL (0.5-1 mg) of re-suspended carbon column material and rinsed three times with 80 µL of water. Oligosaccharides (from 1 to 10 µg dissolved in 80 µL of water) were adsorbed on the graphitized carbon surface. Salts were washed off by rinsing the column three times with 80 µL of water. Organic impurities, such as PEG and Triton X-100, or neutral sugars, were washed off by rinsing the column with 50 µL of 50% methanol in 0.1% TFA (v/v). Finally, sialylated N-glycans were eluted by the application of 20 µL of 50% acetonitrile in 0.1% TFA (v/v). The last fraction was collected and dried in a vacuum or immediately analyzed by nanoESI QTOF in negative ion mode. Tryptic Digest Derivatization. Stabilized rHuEPO was desalted (Microcon YM-10) and reconstituted at a concentration of 1.5 µg/µL in 10 µL of water, and further mixed with 1 µL of Tris acetic buffer (pH 8.5, 1 M), 1 µL of protease trypsin (concentration 1 µg/µL, Merck, Darmstadt, Germany), and 2 µL of water. The mixture was incubated for 18 h at 37 °C. The digestion was stopped by heating at 100 °C (boiling water bath) for 1 min. Then, 1 µL of PNGase F (concentration 1 µg/µL) was added to cleave N-glycans from asparagines. The mixture was incubated for another 18 h at 37 °C. A volume of 9 µL was analyzed for determination of O- and N-glycosylation sites. Finally, a volume of 1 µL of the peptides mixture was diluted in 50 µL of 0.1% TFA and derivatized by 50 µL of 0.8 M 2-methoxy-4,4,5,5-tetradeutero-1H-imidazole (Agilent Technologies, Wilmington, DE) and 0.03 M β-mercaptoethanol. After tagging, a reaction volume of 10 µL was purified using a Cleanup C18 Pipet Tip (Agilent Technologies, Wilmington, DE). The purified and derivatized peptides’ mixture was analyzed by nanoESI QTOF MS. Instrumentation. N-Glycans were analyzed by nanoESI MS and MS/MS using a quadrupole time-of-flight (Q-TOF) mass spectrometer (Waters, Milford, MA) equipped with a nanoESI Journal of Proteome Research • Vol. 5, No. 11, 2006 3067
research articles source. Sialylated N-glycans and derivatized peptides were analyzed in negative ion and positive ion mode, respectively. The glass capillaries were produced with an in-house-made capillary puller. The capillary voltage was set to approximately 1100 V, and the cone voltage was set in the range of 50-70 V. MS/MS analysis of N-glycans was performed in negative ion mode, N-Glycans were fragmented with argon at a pressure (recorded on the instrument’s pressure gauge) of 0.5-0.7 bar; variation of collision energy was in the range from 20 to 50 eV. The instrument conditions for derivatized peptides were the same as for analysis of N-glycans, except that the polarity was positive, cone voltage was 40 V, and collision energy was 60 eV. For the assignment of the N-glycan-derived fragment ions, the general rules established by Domon and Costello25 were followed. O-Glycopeptides and N-deglycosylated tryptic peptides were analyzed by Q-TRAP instrument (AB, Foster City, CA). Polarity was positive, ion spray voltage 5500 V, declustering potential 30 V, and entrance potential 10 V. Collision energy was set to 35-40 eV, and in the MS3 experiment, the excitation time was 150 ms and excitation energy 100 V. MALDI MS analysis of N-glycans was performed on a MALDI-TOF Voyager DE STR Biospectrometry Workstation (AB, Foster City, CA). A mixture of 1.9 mg of 2,4,6-trihydroxyacetophenone and 2.3 mg of ammonium citrate dibasic, THAP/ DAC (Sigma-Aldrich, St. Louis, CA), dissolved in 1 mL of water/ acetonitrile (1:1, v/v), was used as a matrix. The THAP/DAC matrix was earlier found to improve sensitivity of the acidic carbohydrates analysis by significantly contributing to decrease in-source dissociation.7,26 A volume of 1 µL of a solution of N-glycans was mixed with 3 µL of the matrix solution, and 1 µL of this mixture was deposited onto the MALDI plate. Reversed-Phase Chromatography. To separate O-glycopeptides and N-deglycosylated tryptic peptides, a C4 column (GRACE VYDAC, Hesperia, CA,2.1 × 100 mm, pore size 30 nm, and particle size 3 µm) was used. A Q-TRAP instrument connected to an Agilent 1100 Series LC system was used for glycopeptide and peptide structural analysis. The column was equilibrated for 10 min with channel A that contained 0.06% TFA; the peptides were eluted over 115 min with a linear gradient of 0-61% channel B that contained 0.06% TFA diluted in solution of 90% acetonitrile and 10% water (v/v). The flow rate was 0.75 mL min-1.
Results and Discussion Analysis of the Amino Acid Sequence and N-Glycosylation Sites. An overall strategy chosen for the analysis of highly glycosylated proteins is shown in Figure 1. In principle, there are three main analytical directions each targeting characterization of the specific structural elements of glycoprotein. Thus, the strategy covers structural and substructural analysis of glycans, glycopeptides, and deglycosylated peptides with high efficiency and reliability. Each direction and its analytical steps will be explained in more details in their corresponding section. The protein sequence of rHuEPO consists of 165 amino acids and carries three N-glycan chains at Asn24, Asn38, and Asn83. After tryptic or Lys-C digestion, the sequence coverage was 67% because peptides T5 and T9 carrying carbohydrates cannot be correctly assigned. Since PNGase F N-deglycosylated the glycopeptides with the same efficiency as glycoproteins, the dual digestion of glycopeptides released after tryptic or Lys-C digestion was necessary to produce peptides released from N-carbohydrates. Interestingly, only after N-deglycosylation the 3068
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Figure 1. The direct in-solution approach toward elucidating the structure of a glycoprotein.
Figure 2. Amino acid sequence of rHuEPO with tryptic peptides identified in MS (singly underlined) without dual digestion. Doubly underlined peptides represent tryptic peptides identified after deglycosylation. The asterisk marks the asparagines found by MS/MS and MS3 to be N-glycosylated. Triply underlined T13 peptide was identified by MS before dual digestion because 40% of the rHuEPO is not O-glycosylated.6
T5 and T9 peptides became accessible to MS analysis. Consequently, the protein sequence coverage extended to more than 95% (Figure 2). During the process of N-deglycosylation by PNGase F, asparagine converts into aspartic acid, giving rise to a possibility for detection and identification of N-glycosylation sites by MS/ MS or MS3 according to the mass increment gain of 1 Da. Although the LC-MS/MS spectra of the T5 and T9 tryptic fragments represented by triply and doubly charged precursor ions at m/z 897.1 and 787.4, respectively, already revealed the possible N-glycan occupation sites, the MS3 experiment undoubtedly demonstrated and confirmed the exact positions of aspartic acids in both peptides. For the tryptic fragment T5, it was particularly necessary to carry out an MS3 experiment because the intramolecular disulfide bond Cys29-Cys33 hampered the whole peptide sequence analysis (Figure 3). Precursor ions b4+ and y8+, which have the aspartic acid at the C- and N-terminus (m/z 445.1 and 888.3), were extracted from the Q2 and trapped in a Q3 (linear ion trap). When the excitation energy from 100 to 170 V was applied, the b-precursor ions fragmented mostly to b-series ions with neutral loss of water, and the y-precursor ions yielded a y-series of fragment ions. MS/MS sequence elucidation and y-series ion formation of tryptic peptides containing arginine at the C-terminus is usually
Structural Characterization of rHuEPO
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Figure 4. Methylation of cysteine residues that occurs simultaneously with lysine derivatization reaction where An and Ai are amino acid residues closer to the N- or C-terminus, respectively. Figure 3. Posittive ion LC-MS/MS/MS spectra of b4+ and y8+ precursor ions trapped and fragmented by Q3 after the Q2 fragmentation of T5 tryptic peptide ion with intramolecular disulfide bond [EAEDITTGCEAHCSLNEDITVPDTK + H]+ containing N-glycan occupation sites (A) Asn24 and (B) Asn38 converted into the aspartic acid after PNGase F N-deglycosylation.
much easier to achieve than it is for the lysine-containing peptides. Because of the charge distribution of peptide ions in a gas phase, the guanidine or imidazole group placed at the side chain in arginine at the C-terminus will attract most of the protons. Consequently, the ionization efficiency, as well as y-series ion formation, will be approximately 10 times higher relative to the C-terminus lysine-containing peptides.14 To address the issue of ionization efficiency of lysine-containing peptides, a mild tagging reagent, namely, 2-methoxy-4,5dihydro-1H-imidazole, and its deuterated analogues were introduced to the C-terminus lysine.27 Recently, we have shown that a beneficial side reaction of this derivatization is the cysteine methylation (Figure 4).11 This side reaction is induced by the addition of 30 mM β-mercaptoethanol into the reaction mixture. Basically, in a single derivatization reaction of more than 95% reaction efficiency, the intramolecular disulfide bonds were reduced, cysteines were methylated, and lysines were tagged. It was observed that the newly formed peptide containing an imidazole group at the C-terminus lysine became more resilient to the collision energy values (25-40 eV) applied for the underivatized peptides. At elevated collision energy values, that is, 60 eV, the precursor ions started to fragment almost exclusively to y-series ions. The derivatization simplified MS/ MS spectra and helped in unambiguous determination of the peptide sequence (Figure 5). Finally, by this approach, the assignation of aspartic acids converted from asparagines was easier and faster relative to LC-MS/MS28,29 and -MS3 experiments. Noteworthy, by the exclusion of the derivatization step from this method, the detected carbohydrate structures (data not shown) concur with those detected when the derivatization
was involved. Thus, in contrast to many other derivative reagents, the 2-methoxy-4,5-dihydro-1H-imidazole and its deuterated analogues did not change or alter the carbohydrate structures in any way.11 Identification of N-Glycans by MALDI-TOF and ESI-QTOF MS Analysis. MS analysis of N-glycan mixtures obtained by PNGase F digestion of glycoprotein gives arguably superior performance in comparison to analysis of N-glycan-separated fractions. This is mostly related to the sensitivity of analysis, since successive separations leads to sample loss. However, for reliable assessment of the N-glycan variants directly from a mixture, essentially the criteria for high resolution of detection and minimized in-source decay should be concurrently met. To separate and isolate a single N-glycan present in a mixture, two to three chromatographic steps such as anion-exchange,12,28 reversed-phase,29,30 high-pH anion-exchange,31,32 graphitized carbon, and hydrophilic interaction normal phase LC33,34 are generally applied. This approach is time-consuming and requires a few milligrams of the starting material. Therefore, in our study, a direct alternative to the existing protocols for the analysis of N-glycans was considered and developed to offer sensitive and reliable determination of N-glycan heterogeneity. For analysis of the rHuEPO N-glycans at picomole quantities, the desalted N-glycan mixture was submitted to MALDI-TOF MS analysis. In the linear mode option, the intact, sialylated N-glycoforms can bedetected, in contrast with the reflectron mode analysis, by which metastabile or fragment ions are readily formed and detected.6,35,36 Thus, for the linear mode MALDI MS investigation, 50-100 fmol of the purified N-glycan mixture deposited onto the MALDI target allowed the determination of all intact N-glycan species in the mixture. The disadvantage of using a linear analyzer, however, is the low mass precision of detection up to 0.5 Da and the impossibility of carbohydrate ions sequencing. Because of the low resolution and mass precision, the data obtained in the linear mode MALDI MS were used for a rough correlation with the NMR data previously reported,12,13 thus, yielding a useful indication for further structural analysis (Figure 6). Journal of Proteome Research • Vol. 5, No. 11, 2006 3069
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Figure 5. Deconvoluted positive ion mode nanoESI-QTOF-MS/MS spectra of the doubly charged precursor ions at m/z 1396.2 corresponding to T5 tryptic fragment containing derivatized lysine and methylated cysteine. The amount of derivatized tryptic digest in capillary was 10 pmol.
Figure 6. Negative ion linear MALDI-TOF mass spectrum of the N-glycans mixture, containing bi-, tri-, and tetraantennary species, obtained from rHuEPO by N-glycosidase F digestion after purification with graphitized carbon. A total of (2 mg/mL)/10mM THAP/DAC was used as a matrix. Most of the ions were assigned according to the molecular ion information.
To get additional confirmation of the proposed structures, higher mass accuracy and resolution were required. In the reflectron mode, the same ion species could be detected with superior mass accuracy obtained by external calibration up to 0.05 Da and with higher resolution up to 17 000 (Figure 7A). However,, the ion-to-noise ratio was dramatically increased to the level of 10-20% of the most intense ion, at the expense of generation of fragment and metastabile ions. Yet, lowabundance ions related to the glycans N2/D and N4/D were barely detectable, and without the data obtained by the linear mode analysis, it would be hard to achieve correct species assignment. THAP/DAC was used as a matrix for both linear and reflectron measurements showing superior characteristics in comparison to ATT or DHB (6-aza-2-thiothymine or 2,5dihydroxybenzoic acid, respectively), which were found to be more adequate for neutral N-glycans’ analysis. Crucial for the isolation of N-glycans is the purification on the carbon material to adsorb the sugars at the activated 3070
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Figure 7. Negative ion mass spectra of rHuEPO N-glycans comprising bi-, tri-, and tetraantennary structures. The major ions are assigned to the proposed sugar structures. (A) Singly charged N-glycans obtained by MALDI-TOF in the reflectron mode, THAP/ DAC [(2 mg/mL)/10mM] was used as a matrix. (B) Multiply charged N-glycan ions obtained by nanoESI QTOF MS in solution of 1:1 MeCN-H2O (v/v).
surface,7,24 working even under heavy pollution with PEG or Triton 80. Since rHuEPO contains only sialylated N-glycans, particular attention was paid toward this structural element
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Structural Characterization of rHuEPO Table 1. Calculated and Obtained Monoisotopic and Average m/z Values of rHuEPO N-Glycans
N-glycans
[M - H]calculated average
calculated monoisopic m/z
[M - H]m/z
N1/A N2/A
2077.9001 2369.1518
2077.7455 2368.8409
2076.7377 2367.8331
N2/B N3/B
2734.4954 3025.7533
2733.9731 3025.0685
2732.9653 3024.0607
N2/C N3/B N2/D N4/A N3/C N4/B N3/E N4/C N4/D
3099.8328 3391.0907 3465.1701 3682.3486 3756.4281 4047.6860 4121.7654 4413.0233 4778.3607
3099.1052 3390.2007 3464.2375 3681.2961 3755.3329 4046.4283 4120.4651 4411.5605 4776.6927
3098.0975 3389.1929 3463.2297 3680.2883 3754.3251 4045.4205 4119.4573 4410.5527 4775.6849
[M - 2H]2m/z
[M - 3H]3m/z
Biantennary 1037.8649 691.5740 1183.4126 788.6058 Triantennary 1365.9787 910.3165 1511.5264 1007.3483 Tetraantennary 1548.5448 1032.0273 1694.0925 1129.0591 1731.1103 1153.7380 1839.6402 1226.0909 1876.6584 1250.7698 2022.2063 1347.8016 2059.2247 1372.4805 2204.7724 1469.5123 2387.3385 1591.2231
which highly influences the adsorption on the surface of carbon particles through the polar interactions with TFA. This behavior is more prominently exhibited by acidic carbohydrates than by the neutral ones. An additional rinsing step with methanol, which is less polar than acetonitrile, will elute from the column only organic impurities (like glycerol, remained proteins, etc.) or neutral sugars. Inorganic impurities were eluted from the column in earlier rinsing steps with water. That was additionally confirmed by two experiments: first, by analyzing the methanol fractions on the anion-exchange column and HPAEC-PAD instrument, and second, by mixing two erythropoietin N-glycan mixtures, N-glycans with sialic acids and desialylated N-glycans produced by enzymatic digestion with NANase II.37 In the first analysis, the eluted carbohydrates were not detected in methanol fractions, in contrast to the second analysis where only neutral sugars in methanol (data not shown) and sialylated ones in acetonitrile fractions (Figure 7) could be found. Following the optimization of the N-glycans purification procedure, the N-glycan fractions contained in acetonitrile were subjected to nanoESI-QTOF MS in the negative ion mode. All previously identified N-glycan variants, with the exception of N1/A (Figure 7B) were detected with high mass accuracy and resolution. Summarized results are presented in Table 1. As revealed by both MALDI and ESI MS analysis, the N-glycan mixture is mostly dominated by tetraantennary and triantennary structures. This could give an indication about in vivo activity of this batch of rHuEPO, since a positive correlation between number of antennae and in vivo activity has been already demonstrated.38 Another peculiar feature arising from the spectra in Figures 6 and 7 is the presence of the tetrasialylated N-glycans at reasonable or high S/N. Careful adjustment of the MS ionization parameters to maintain the sialic acid attached is of particular importance to define finally the pharmaceutical quality control and use of the rHuEPO. It is known that patients with anemia express sera EPO that lacks tetra-acidic N-glycans.39 In this context, it can be envisaged that such a well-defined MS protocol may reveal glycan structural discrepancies between human serum EPO and rHuEPO for diagnosing pathological conditions,39 and/or the presence/ absence of tetrasialylation in urinary hEPO.38 Only with a full set of data obtained with the high mass accuracy and resolution reflectron instruments (MALDI-TOF or ESI QTOF) complemented with the linear mode analysis can the correct assignment of the N-glycan species be achieved.
[M - 4H]4m/z
obtained monoisopic m/z nanoESI MS
obtained monoisopic m/z MALDI-TOF
obtained average m/z MALDI-TOF
518.4285 591.2024
/ 1183.4244
2076.7735 2367.7735
2077.9 2369.2
682.4854 755.2593
1366.0162 1511.6245
2733.1005 3024.0996
2734.6 3025.9
773.7685 846.5423 865.05015 919.3162 937.8254 101.5992 1029.1084 1101.7723 1193.1653
1548.6229 1694.1589 1731.1602 1226.0918 1250.7660 1347.8153 1372.5028 1469.5662 1193.1958
3098.1117 3389.1875 3463.2051 3680.2774 3754.2917 4045.4201 4119.4987 4410.5832 4775.7149
3100.2 3391.3 3465.6 3682.5 3756.6 4047.6 4121.8 4413.5 4778.8
Identification and Structural Analysis of O-Glycans. To identify the O-glycans in the tryptic mixture, a dual digestion approach was carried out. In the first step, the protease digestion by trypsin cleaves rHuEPO into peptides followed by the release of N-glycans from the peptides by endoglycosidase PNGase F. The second digestion is crucial for discrimination between O-glycopeptides and N-glycopeptides, since according to the chromatograms they are coeluting at the same retention time of 45-47 min (Figure 8). Carbohydrate fragment ions of low m/z values emerging at low collision energies in complex TIC chromatograms of protease digests can be detected and subsequently identified as O-glycosylation-related diagnostic ions, such as m/z 366 Hex-HexNAc+, m/z 292 Neu5Ac+, m/z 204 HexNAc+, m/z 163 Hex+, and so forth. This procedure can be successfully applied only if N-glycans were removed by PNGase F in dual digestion mode.40 By extracting the diagnostic ions from the TIC chromatogram the target O-glycan, ions can be recognized, identified, selected as precursor ions, and sequenced for detailed structural determination. This methodology will be explained in details in the O-glycans section. Thus, after the N-glycans release, only the O-glycopeptides remained in the peptide mixture, giving the opportunity to be easily detected by performing extracted ion chromatograms within the mass window of 1 Da for the m/z values 204 (HexNAc+), 292 (Neu5Ac+), 366 (Hex-HexNAc+), and 657, (NeuAc-Hex-HexNAc+), respectively, characteristic for O-glycans. The lack of an O-glycosidase for detachment of all glycans linked to Ser/Thr and severe conditions for the classical chemical cleavage by β-elimination in basic environment causing peptide degradation22,41,42 usually make the investigation of O-glycosylation more difficult,43,44 and frequently, incomplete data are obtained. As an alternative, the direct MS analysis of O-linked peptides is a more efficient analytical tool. By our approach within a single LC-MS and MS/MS experiment of the dual digestion mixture, the O-glycans were identified and sequenced, and the position of glycosylation sites of both N- and O-glycans was unambiguously determined. Both T13 O-linked glycans detected within the elution time from 46.0 to 48.6 min were fragmented by MS/MS. The highest peptide sequence coverage as well as a successful differentiation between the trisaccharide- and the tetrasaccharidecontaining peptides was achieved by the triple quadrupole instrument (Q-TRAP). The trapping options can be well-controlled, such as the prolonged accumulation time Journal of Proteome Research • Vol. 5, No. 11, 2006 3071
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Figure 8. Overlaid UV/vis (214 nm) chromatograms of rHuEPO after digestion by trypsin (upper trace) and after dual digestion by trypsin and PNGase F (lower trace). The conversions of asparagines into aspartic acids indicating the N-glycosylation sites at the amino acid D are marked with an asterisk. Table 2. Fragment Masses of T13 Tryptic rHuEPO Peptide Contain O-Linked Glycans Neu5AcR2-3Galβ1-3GalNAc and Neu5AcR2-3Galβ1-3(Neu5AcR2-6)GalNAc Obtained by LC-MS/MS Analysis amino acid sequence T13
yn
bm
R
1
15
y1+(175.12)
glycoprotein or protein ion (calculated m/z)
T13-Ser126GaINAcGal (NeuAc)2 obtained m/z
T13-Ser126GaINAcGalNeuAc obtained m/z
/
175.17
1447.77
1447.77
L
2
14
b14+(1447.76)
P
3
13
y3+(385.26)
385.23
385.29
A
4
12
y4+(456.20)
456.27
456.27
A
5
11
b11+(1010.48)
1010.37
1010.55
S
6
10
y6+(614.36)
614.49
614.49
A
7
9
A
8
8
D P
9 10
7 6
P
11
5
S
12
4
I A
13 14
3 2
E
15
1
685.41 888.57 756.39 959.43 871.47 968.55 1171.65 533.37 634.89 1065.57 1268.67 1152.57 1355.61 1265.51 1336.61 / 733.71 834.99 980.49 1465.65 1668.87
685.41 888.57 756.45 959.43 871.47 968.49 1171.53 533.43 634.95 1065.57 1268.61 1152.63 1355.73 1265.55 / 288.09 733.47 835.05 / 1465.65 1668.87
y7+(685.40) y7-Ga1NAc+(888.48) y8+(756.44) y8-Gal1NAc+(959.52) y9+(871.46) y10+(968.52) y10-Ga1NAc+(1171.60) y112+(533.29) y11)Ga1NAc2+(634.83) y11+(1065.57) y11-Ga1NAc+(1268.65) y12+(1152.60) y12-Ga1NAc)(1355.68) y13+(1265.68) y14+(1336.72) b2+(288.20) T132+(733.77) T13-Ga1NAcNeuAc2+(834.93) T13-Ga1NAcNeuAc2+(980.48) T13+(1465.77) T13-Ga1NAc+(1669.84)
of the doubly charged precursor ions, m/z 1061.50 for the trisaccharide-containing paptide and m/z 1207.05 for the tetrasaccharide-containing peptide, as well as ramping of the collision energy. The two serines found in the peptide sequence EAISPPDAASAAPR of both O-glycopeptides (117-131 residue of rHuEPO) can be considered as potential O-glycans linkage sites: Ser-120 and Ser-126. The serine at the position 120 can be excluded as the possible O-glycan occupation site by the 3072
Journal of Proteome Research • Vol. 5, No. 11, 2006
carbohydrate ion m/z
GaINAcGal(NeuAc)2 m/z
GaINAcGalNeuAc obtained m/z
Ga1+ (163.06) Ga1NAc+ (204.09) NeuAc+-H2O (274.09) NeuAc+ (292.10) Ga1NAcNeuAc+ (366.14) Ga1NAcNeuAc+ (495.18) Ga1NAcGa1NeuAc+ (657.24) Ga1NAcGa1NeuAc2+ (948.33)
/
163.08
204.15
204.21
274.17
274.11
292.17
292.17
366.09
366.21
495.28
/
657.21
657.24
948.33
/
presence in MS/MS spectra of the y7-GalNAc+ ions along with the entire series of N-acetylhexosamine-containing ions from y11-GalNAc+ to y8-GalNAc+ (Table 2). In the MS/MS of the tetrasaccharide-containing peptide ions, the GalGalNAc (m/z 366.21), GalNAcNeu5Ac+ (m/z 495.28), and GalNAcGalNeu5Ac2+ (m/z 948.33) are indicative for the core type 1. In addition, the ion at m/z 308.11 clearly supports the presence of Neu5AcR26GalNAc linkage.45 GalNAcNeu5Ac+ and a corresponding ring
Structural Characterization of rHuEPO
research articles
Figure 9. MS/MS spectra of O-linked glycans identified in the TIC chromatogram after dual digestion and fragmented by MS/MS. (A) Tryptic fragment T13 contains Neu5AcR2-3Galβ1-3GalNAc; precursor ion was the doubly charged m/z 1061.50. (B) Tryptic fragment T13 contains Neu5AcR2-3Galβ1-3(Neu5AcR2-6)GalNAc; precursor ion was the doubly charged m/z 1207.05.
cleavage at nominal m/z 30845 were not found in the MS/MS spectra of the trisaccharide-containing peptide, proving that the single sialic acid moiety is linked to galactose by a 2-3 linkage. This finding is in agreement with the characteristic of the CHO cells to lack a sialyl R2-6 transferase.46 The proposed structure of the tetrasaccharide-containing peptide was additionally confirmed by the doubly charged ion T13GalNAcNeu5Ac2+. The linkage between galactose and sialic acid was confirmed by the GalNeu5Ac+ ions obtained in both MS/ MS spectra (Figure 9). Additionally, to assess a possible presence of an unglycosylated T-13 peptide in rHuEPO, the peak at retention time 49.2 was submitted to the MS/MS experiment selecting as a precursor ion the doubly charged m/z 733.39. By analyzing all three MS/MS spectra, the presence and structure of the tetrasaccharide carrying two sialic acid moieties, the linear trisaccharide comprising one sialic acid residue, as well as the unglycosylated rHuEPO, could be straightforwardly determined. After deglycosylation by PNGase F, the protein precipitated after detaching N-glycans. Protein left after sugar extraction was analyzed by MALDI-TOF in linear ion mode. In this experiment, the same three structures postulated previously: tetrasaccharide and trisaccharide O-glycans and non-
O-glycosylated rHuEPO were detected under mass accuracy of (5 Da at m/z 19190.9, 18898.3, and 18240.1, respectively.47 Structural Analysis of N-Glycans. The main interest in analysis of the N-glycan mixtures was to find diagnostic ions for differentiation between fully sialylated antennae and those lacking a sialic acid moiety. MS/MS analysis has been carried out on the N-glycan mixtures purified on the graphitized carbon column using the same procedure described for nanoESI MS and MALDI-TOF analysis. Triply charged precursor ions at m/z 1129.06 and 1226.09 detected in the negative ion mode (N3/B and N4/A) were selected and fragmented to deliver mostly doubly charged ions in the mass region from m/z 1200 to 1500. These ions represent a combination between cross-ring cleavages and internal fragments formed by cleavages of glycosidic bonds.48 The position of the fucose unit was determined by calculation of the mass difference between the ions Y6R′,R′′,β′ and B6/Y6R′,R′′,β′ or Z6R′,R′′,β′ and B6/Y6R′,R′′,β′. Additional confirmation of the Fuc location was obtained through the fragment ions generated by neutral loss of sialic acid moiety and ring cleavage at the HexNAc residue at the reducing end. These are 0,2A7/ Y6R′,R′′,β′ or 0,2A7/Z6R′,R′′,β′, 2,4A7/Y6R′,R′′,β′, and 2,4A7/Z6R′,R′′,β′ where the pattern of Y and Z ion formation in pairs with an m/z difference Journal of Proteome Research • Vol. 5, No. 11, 2006 3073
research articles
Cindric´ et al.
Figure 10. Negative MS/MS spectra of triply charged [M - 3H]3- trisialylated N/3B carbohydrate ion at m/z 1129.1.
of 18 also exists. On the other hand, no other fragment ion that would support nonfucosylated or other Fuc position,49 that is, a Neu5AcHexHexNAcFuc or Neu5AcHexHexNAcHexFuc, could be detected. This fucosylation pattern is in agreement with the one expected to be in EPO-CHO under standard conditions, since these cells do not express 1-3/4 fucosyl transferases.50-52 B-ions after loss of two sialic acids such as B6/Y6R′,R′′,β′/Y6R′,R′′,β′ or ions 2,4A6/Y6R′,R′′,β, 0,2A6/Y6R′,R′′,β are not appropriate for differentiation between N3/B and N4/A. The diagnostic ion common for all similar structures in rHuEPO carbohydrates found and assigned as D-ion (B5/Y3β) at m/z 817.29 provided the information of an antennae.7 A similar ion like B5/Y3R that could point out the difference between N3/B and N4/A N-glycans was not found. The triply charged ions formed from the precursor ion and cross-ring cleavages of 2,4A7 and 0,2A7 in the m/z region from 1000 to 1200 containing their sialic acid moieties are diagnostic for differences between tri- and tetrasialylated tetraantennary glycans. The only difference found by comparing two MS/MS spectra that defined a branch without the sialic acid was the presence of the singly charged ion C4β which was derived by the cleavage of the antennae specific for N3/B (Figure 10). Another difference found in the N3/B MS/ MS spectrum was a higher signal intensity of the ions at m/z 364.12 and 382.14. Those ions could be generated after cleavage at the positions B2β′′ or C2β′′ or by double cleavage of the glycosidic bond as B3β′/Y6β′, B3R′/Y6R′, and B3R′′/Y6R′′ and C3β′/Y6β′, C3R′/Y6R′, and C3R′′/Y6R′′. However, the generation of disaccharide ions by single glycosidic bond cleavage is more favored than 3074
Journal of Proteome Research • Vol. 5, No. 11, 2006
by internal double cleavage; therefore, it might be considered as an indication for N3B versus N4A presence. The most structural data in negative ion mode were obtained by glycosidic cleavages between the residues of the chitobiose core. Those data in combination with signal intensity data of singly charged ions can reveal small structural differences between N-glycans. As indicated in Figure 10, R′, R′′, β′, and β′′ stand for the various branches of the structure. The comma separating a nomenclature like Y6R′,R′′,β, stands for “and/or”.25 Basically, either of these cleavages and/or simultaneous such cleavages could occur, each rendering fragment ions of the same m/z value. Only the typical structural elements for the CHO-produced rHuEPO were observed to differ from human natural EPO: the presence of Neu5AcR2-3Gal versus Neu5AcR2-6Gal, no bisecting N-acetyl glucosamine could be detected/deduced, no position of the Fuc other than at the core GlcNAc (Fuc 1-6GlcNAc) was detected, a slightly increased number of LacNAc repeat per molecule.38,39,46,50 Otherwise, the glycosylation profile is similar to that of human EPO, documenting the amenability of this rHuEPO batch for pharmaceutical purposes. Thus, the potential of the present method to offer deep and numerous insights into glycoprotein structure is highlighted.
Conclusions The present study describes a new approach for MS-based investigation of rHuEPO N- and O-glycosylation status at the
research articles
Structural Characterization of rHuEPO
low picomole level. It consists of derivatization by 2-methoxy4,5-dihydro-1H-imidazole of the peptides and O-glycosylated peptides released after dual digestion of rHuEPO by trypsin and PNGase F, and subsequent analysis by LC-MS and MS/MS. This derivatization process gives rise to two essential effects upon MS analysis of dually digested rHuEPO: (a) increased ionization efficiency of the derivatized lysinecontaining peptides and O-glycosylated peptides and (b) and the preferential generation of y-series of fragment ions. The increased ionization efficiency of the lysine-containing peptides and O-glycopeptides facilitated the coverage of the protein sequence up to 95%. In the same manner, the lysine-containing peptides comprising also the N-glycosylation sites exhibited superior ion yield, thus, enabling their extensive MS and MS3 analysis, with predominant formation of the y-series of fragment ions. Consequently, the N-glycosylation sites of the rHuEPO could be unambiguously defined by MS and MS3 analysis. A beneficial peculiarity of this derivatization method resides in the fact that the structures of O-glycans attached to peptides are not altered to any extent, yet giving rise to elevated ion yield of O-glycosylated peptides. This enhances on one side their detection by MS, and their structural characterization by MS/MS on the other. By the selection in LC-MS mode of the indicative ions for the presence of O-glycans and further MS/ MS analysis of the corresponding precursor ions, the Oglycopeptides could be straightforwardly identified and structurally defined. Thus, complete characterization of the peptide chain, the clear-cut location of O-glycosylation, and a full set of fingerprint ions for structural architecture of O-glycans could be achieved. Basically, in single LC-MS and MS/MS experiments, three structural features of the glycoprotein could be reliably determined: the protein sequence, the location of the N-glycosylation sites and O-glycosylation site, and the corresponding glycan variants. For the N-glycans analysis, a simplified protocol has been developed, encompassing only one single purification step, namely, graphitized carbon column followed by MS analysis. The compositional mapping of N-glycans mixture released after N-deglycosylation carried out by MALDI MS in linear mode provides intact molecular ion species, while high resolution and accurate detection of N-glycan ions species could be achieved by reflectron mode. Subsequently, in this context, a highly sensitive analysis of the N-glycans mixture heterogeneity could be reliably assessed. The complete structural investigation of the N-glycan variants was achieved by tandem MS analysis. Additionally, the possibility to define characteristic structural elements for discrimination between fully sialylated and partially desialylated antennary N-glycans was explored. An overview of possible directions toward elucidating the structure of a glycoprotein is given in Figure 1. Despite the multi-methodological feature of this approach, it basically represents a simple, efficient, and fast alternative for glycoprotein analysis that allows complete characterization of Oand N-glycosylation sites and status, as well as protein sequence. Its potential as a general analytical protocol in glycoproteomics is highlighted.
Acknowledgment. ‘The authors are grateful to Pliva Research & Development Ltd. The ESI Q-TOF instrument was provided by HbfG grant (Land Nordrhein-Westfalen, Germany) to Jasna Peter-Katalinic´.
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PR060177D
