Characterization of Oligosaccharides in Recombinant Tissue

Anal. Chem. 2009, 81, 9744–9754

Characterization of Oligosaccharides in Recombinant Tissue Plasminogen Activator Produced in Chinese Hamster Ovary Cells: Two Decades of Analytical Technology Development Oleg V. Borisov,* Matthew Field, Victor T. Ling, and Reed J. Harris Protein Analytical Chemistry Department, Genentech, Incorporated, 1 DNA Way, South San Francisco, California 94080 Recombinant tissue plasminogen activator (rt-PA) is a well-characterized glycoprotein with a great deal of published information on its structure, post-translational modifications, and O- and N-glycosylation. Most of the characterization was accomplished in the late 1980s. During the past 2 decades, however, mass spectrometry has made a quantum leap forward offering new capabilities in soft electrospray ionization, speed, resolution, and accuracy of mass measurements. From this point of view, it is worthwhile to revisit the characterization of familiar proteins, such as rt-PA, using the new capabilities of modern analytical technology. In this work, we applied LC-MS with state-of-the-art instrumentation to the characterization of glycoforms of rt-PA. This method takes advantage of accurate mass measurements along with a fast “in-source” voltage switching for the detection of characteristic oxonium ions of saccharides. This method confirmed previously identified glycan structures based on existing knowledge of rt-PA glycans. In addition, we identified two novel glycan structures in rt-PA. A low level of Asn142 N-glycosylation was detected at an atypical Asn-Xaa-Cys consensus motif. It was found to be modified predominantly by biantennary hybrid structures. This N-glycosylation site was confirmed using a recently developed electron-transfer dissociation (ETD) technique. Also using this method, we detected low levels of elongation of fucose-O-Thr61 to di-, tri-, and tetrasaccharides, not previously observed in rt-PA. The results demonstrate that use of state-of-the-art analytical methods can reveal low-level, previously undetected modifications of well-characterized biopharmaceuticals. Within the last 2 decades significant progress has been made in technologies for the structural elucidation and characterization of biological molecules, mainly due to the development of “soft” sample introduction methods, such as electrospray ionization1 and advances in mass spectrometry (MS) in general. Modern mass spectrometers offer high sensitivity, resolution, and mass accuracy on a time scale to match chromatographic separations. From the * To whom correspondence should be addressed. E-mail: [email protected]. (1) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64–71.

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perspective of recent advances in bioanalytical technology, it is interesting to revisit the analysis of a familiar sample. The application of mass spectrometry to the analysis of glycosylation of proteins and peptides and identification of novel N-linked oligosaccharides has grown in the recent years.2-4 Glycoprotein rt-PA is a serine protease, converting plasminogen into plasmin, which has important thrombolytic properties due to its high fibrin affinity.5 It was first approved by the FDA in 1987 for treating acute myocardial infarction, or heart attacks, and later for acute ischemic stroke, and acute, massive pulmonary embolism. rt-PA can be produced by recombinant DNA technology in Chinese hamster ovary (CHO) cell lines. Its amino acid sequence consists of 527 residues. Production of rt-PA in mammalian cell lines results in a high degree of glycosylation, which can be the source of microheterogeneity in rt-PA. Three major N-linked sites and one O-linked site have been previously identified. Site-specific N-glycosylation at typical Asn-Xaa-Ser/Thr motifs are due to asparagine glycosylation at sequence positions 117, 184, and 448.6-11 Glycosylation at each individual site is heterogeneous: Asn117 glycosylation site contains high-mannose structures, whereas Asn184 and Asn448 are modified by complex/ hybrid oligosaccharide structures. Although certain N-glycosylation features are conserved irrespective of the production cell type used for t-PA expression, the relative incidence of common structures is unique to each of the cell types.12 For (2) Harvey, D. J. Expert Rev. Proteomics 2005, 2, 87–101. (3) Takahashi, N.; Masuda, K.; Hiraki, K.; Yoshihara, K.; Huang, H. H.; Khoo, K. H.; Kato, K. Eur. J. Biochem. 2003, 270, 2627–2632. (4) Takegawa, Y.; Deguchi, K.; Nakagawa, H.; Nishimura, S. Anal. Chem. 2005, 77, 6062–6068. (5) Collen, D.; Stump, D. C.; Gold, H. K. Annu. Rev. Med. 1988, 39, 405–423. (6) Vehar, G. A.; Spellman, M. W.; Keyt, B. A.; Ferguson, C. K.; Keck, R. G.; Chloupek, R. C.; Harris, R. J.; Bennett, W. F.; Builder, S. E.; Hancock, W. S. Cold Spring Harbor Symp. Quant. Biol. 1986, 51, 551–562. (7) Carr, S. A.; Roberts, G. D.; Jurewicz, A.; Frederick, B. Biochimie 1988, 70, 1445–1454. (8) Spellman, M. W.; Basa, L. J.; Leonard, C. K.; Chakel, J. H.; O’Connor, J. V.; Wilson, S.; van Halbeek, H. J. Biol. Chem. 1989, 264, 14100–14111. (9) Ling, V.; Guzzetta, A. W.; Canova-Davis, E.; Stults, J. T.; Hancock, W. S.; Covey, T. R.; Shushan, B. I. Anal. Chem. 1991, 63, 2909–2915. (10) Pohl, G.; Ka¨llstro ¨m, M.; Bergsdorf, N.; Walle´n, P.; Jo ¨rnvall, H. Biochemistry 1984, 23, 701–707. (11) Vehar, G. A.; Kohr, W. J.; Bennett, W. F.; Pennica, D.; Ward, C. A.; Harkins, R. N.; Collen, D. Biotechnology 1984, 2, 1051–1057. (12) Parekh, R. B.; Dwek, R. A.; Rudd, P. M.; Thomas, J. R.; Rademacher, T. W.; Warren, T.; Wun, T.-C.; Hebert, B.; Reitz, B.; Palmier, M.; Ramabhadran, T.; Tiemeier, D. C. Biochemistry 1989, 28, 7670–7679. 10.1021/ac901498k CCC: $40.75  2009 American Chemical Society Published on Web 11/10/2009

example, depending on occupancy at the Asn184 site, type I and II variants are defined. Type I rt-PA is fully glycosylated, whereas type II is completely lacking oligosaccharides at this site. Under typical manufacturing conditions, rt-PA production in CHO cell lines results in approximately 1:1 ratios of type I:II variants. More recently, Harris et al.13 identified an O-fucosylation site in rt-PA, located within the epidermal growth factor (EGF) domain at Thr61. In general, O-fucosylation of Thr/ Ser residues has been reported to occur at conserved sites within -Cys-Xaa-Xaa-Gly-Gly-Thr/Ser-Cys- sequences in epidermal growth factor homology regions (EGF modules) of several multidomain proteins.14,15 In 1998 O’Connor published a historical review on the analysis and characterization of a well-characterized protein, rt-PA.16 In our current study we revisited the characterization of glycans in rt-PA. We report on the application of liquid chromatography-tandem mass spectrometry (LC-MS) to glycosylation profiling of rt-PA expressed in CHO cells. Identification of N-glycosylation is based on examining of reconstructed ion chromatograms (RICs) for the presence of characteristic oxonium ions generated during mild “in-source” collision-induced dissociation (CID) experiments.17,18 In our experience, not only was this method successfully applied toward fast glycan profiling (mapping), which is potentially useful for sample comparability and lot-to-lot screening purposes, but the sensitivity and resolution of the current technology has allowed us to detect the presence of novel glycans in such a well-characterized protein as rt-PA. Further characterization of a novel N-glycosylation site was accomplished by applying recently developed electron-transfer dissociation (ETD)19 methods during online LC-MS analysis. EXPERIMENTAL SECTION Materials. Recombinant DNA derived rt-PA was produced in CHO cell lines and purified in-house (Genentech Inc., South San Francisco, CA). Dithiothreitol (DTT), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris), and iodoacetic acid (IAA) were purchased from Sigma-Aldrich (St. Louis, MO). All solvents including water and acetonitrile were HPLC grade and were purchased from Burdick and Jackson (Muskegon, MI). Trifluoroacetic acid (TFA) and guanidine hydrochloride (GdnHCl) were from Pierce (Rockford, IL). Trypsin, modified sequencing grade, was purchased from Roche (Indianapolis, IN). Tryptic Digest Sample Preparation. An aliquot containing 1 mg of rt-PA was diluted to 1 mg/mL with reduction/carboxymethylation (RCM) buffer (6 M GdnHCl, 35 mM Tris, and 20 mM DTT, pH 7.5) and incubated at 37 °C for 30 min in a water bath. A freshly prepared IAA solution was added to a final concentration of 55 mM, and the carboxymethylation reaction was carried out at 37 °C in a water bath for 30 min in the dark and (13) Harris, R. J.; Leonard, C. K.; Guzzetta, A. W.; Spellman, M. W. Biochemistry 1991, 30, 2311–2314. (14) Harris, R. J.; Ling, V. T.; Spellman, M. W. J. Biol. Chem. 1992, 267, 5102– 5107. (15) Harris, R. J.; Spellman, M. W. Glycobiology 1993, 3, 219–224. (16) O’Connor, J. V. Dev. Biol. Stand. 1998, 96, 113–121. (17) Carr, S. A.; Huddleston, M. J.; Bean, M. F. Protein Sci. 1993, 2, 183–196. (18) Medzihradszky, K. F. Methods Mol. Biol. 2008, 446, 293–316. (19) Syka, J. E.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9528–9533.

quenched by addition of 1 M DTT. The RCM sample was bufferexchanged into digestion buffer (10 mM ACES, 20 mM CaCl2, pH 7.0) using a PD-10 desalting column (GE Healthcare BioSciences, Piscataway, NJ). Digestion was performed by addition of 20 µL of 1 mg/mL trypsin solution followed by incubation at 37 °C for 4 h. Digestion was stopped by addition of 20 µL of 10% TFA solution. Digested samples were stored at 2-8 °C prior to analysis. HPLC Separation of Tryptic Digests. Reversed-phase (RP) separation of tryptic peptides was performed on an Agilent 1100 series high-performance liquid chromatography (HPLC) system (Santa Clara, CA) equipped with UV diode array detector. An Agilent Zorbax 300SB-C18 2.1 mm × 150 mm column packed with 300 Å pore-sized 5 µm particles was operated at 45 °C and a flow rate of 0.400 mL/min. At initial condition and during sample loading, the column was held at 97% of mobile phase (MP) A (0.1% TFA in water) and 3% of MP B (0.08% TFA in acetonitrile). The analytical gradient consisted of a linear ramp from 3% to 30% MP B in 80 min, followed by a ramp to 65% MP B in 25 min, then a 1 min ramp to 3% MP B, and finally equilibration at initial conditions for 30 min. Typically, 35 µg of rt-PA digest was loaded onto the column. The HPLC eluent flow after 1:1 splitting was directed online into a mass spectrometer. Mass Spectrometry and Data Analysis. Glycopeptide profiling was performed using an LTQ Orbitrap XL instrument (Thermo Fisher Scientific, San Jose, CA) operated in a positive ionization mode. The instrument was operated at a nominal resolving power of 15 000 at m/z 400 with typical mass accuracies of better than 5 ppm after external calibration. Two modes of operation were used for glycoprofiling with the first scan event performed in a full-scan MS mode for the range of m/z 200-2000 amu, followed by the second full-scan event acquired at an increased “in-source” fragmentation condition. “In-source” fragmentation was achieved in the skimmer-multipole stack region by offsetting potentials of all multipole elements by 25-30 V compared to their normally tuned settings. These two scan events were alternated throughout the LC-MS run. “In-source” fragmentation data was reconstructed for m/z 204.09, 292.10, and 366.14 characteristic ions, respectively, for oxonium ions of N-acetylhexosamine, N-acetyl-neuraminic (sialic) acid, and hexosyl-N-acetylhexosamine. First, RICs for the oxonium ions were used to detect elution regions of glycopeptides. Then, full-scan MS spectra from the first scan event were averaged over the range of detected oxonium ions and masses of detected glycoforms were matched, with a given mass tolerance, to the theoretical masses of in-silico digested rt-PA peptides, modified by common oligosaccharide structures, typically observed in CHO expressed glycoproteins. Finally, unless otherwise specified, RICs for the most abundant charge states of these structures were used to determine relative amounts of glycans at each site. Characterization of a novel N-glycosylation site at Asn142 was accomplished by ETD fragmentation during online LC-MS experiments. An LTQ XL (Thermo Fisher Scientific, San Jose, CA) equipped with an ETD source was used in this study. In ETD, fluoroanthene radical-anions, generated by chemical ionization, are allowed to react with multiply charged ions of analyte inside an ion trap, resulting in transfer of electrons onto ions of analyte which in turn leads to radical-induced homolytic cleavages at peptide backbones. ETD source parameters were Analytical Chemistry, Vol. 81, No. 23, December 1, 2009

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Figure 1. Reversed-phase LC-MS tryptic peptide map of rt-PA expressed in CHO cell-lines. Total ion current (TIC) chromatogram (a), obtained at normal “in-source” potential and reconstructed ion chromatograms (RICs) for oxonium characteristic ions acquired at elevated “in-source” conditions offset by 25 V due to hexosyl-N-acetylhexosamine (Hex-HexNAc) at m/z 366.14 (b), N-acetylhexosamine (HexNAc) at m/z 204.09 (c), and sialic (NANA) acid at m/z 292.10 (d). Major peaks in RICs are due to glycosylation of T45, T13, T17, T11, and T11a peptides. The minor peak at 67 min corresponds to the O-linked glycopeptide T8 (see text). Mass tolerance of RICs (b-d) was set at 5 ppm.

optimized automatically to achieve fluoroanthene ion counts around 2-5 × 106 counts. During chromatographic separation of rt-PA digest, the LTQ was operated in a full-scan MS mode. In regions where glycopeptides of interest elute, ETD MS2 scan events for triply charged and doubly charged precursor ions were added. ETD reaction time was optimized at 115 ms, and ETD spectra from m/z 100 to 3200 were collected. Supplemental CID was employed during ETD of doubly charged precursor ions in order to improve their fragmentation efficiency. Operation of LTQ XL and LTQ Orbitrap XL instruments and data processing were accomplished with XCalibur (Thermo Fisher Scientific, San Jose, CA) software. Deconvolution of multiply charged spectra was performed with Xtract function of the Qual Browser software. RESULTS AND DISCUSSION LC-MS “In-Source” CID for rt-PA Glycoprofiling. The method for online mapping of glycopeptides utilizes alternating high and low “in-source” potentials. This approach was pioneered by Carr et al.17 and has been recently reviewed in great detail by Medzihradszky.18 During the first scan event acquired at low “in9746


source” potential, masses of peptides, including glycopeptides, are measured. Following this, mildly elevated “in-source” voltage of the second scan event causes partial fragmentation, especially due to cleavages of labile bonds such as glycosidic linkages. Since most of the glycans previously identified in rt-PA typically are either complex/hybrid or high-mannose structures,7-11 oxonium characteristic ions due to N-acetylhexosamine (HexNAc), sialic acid (NANA), and hexosyl-N-acetylhexosamine (Hex-HexNAc) with m/z 204.09, 292.10, and 366.14, respectively, were chosen for this study. Figure 1 demonstrates a LC-MS total ion current (TIC) chromatogram obtained at low “in-source” potential (Figure 1a) and corresponding RICs for the above characteristic ions acquired at elevated “in-source” conditions (Figure 1b-d). As can be noted, “in-source” fragmentation effectively identifies elution regions of glycopeptides, greatly simplifying their detection and characterization. Examination of elution profiles of the characteristic ions suggests the presence of five major glycopeptides. Earlier studies, however, reported three major N-glycosylation sites in rt-PA, namely, Asn117, Asn184, and Asn448.6-11 It has been also shown that digestion with trypsin typically generates four major

Table 1. N-Linked Tryptic Glycopeptides Detected in rt-PA by LC-MS site

peptide

residues

sequon

M+H

elution range, min

sequence

Asn117 Asn117 Asn142a Asn184 Asn448

T11 T11a T13 T17 T45

102-129 102-126 136-145 163-189 441-449

Asn-Ser-Ser Asn-Ser-Ser Asn-Tyr-Cys Asn-Gly-Ser Asn-Arg-Thr

3017.33 2717.18 1204.55 3076.12 1129.54

44.5-46.0 48.0-49.0 26.5-29.0 42.0-44.5 18.5-22.0

GTWSTAESGAECTNWNSSALAQKPYSGR GTWSTAESGAECTNWNSSALAQKPY LGLGNHNYCR YSSEFCSTPACSEGNSDCYFGNGSAYR CTSQHLLNR

a

Previously unreported N-glycosylation site identified in this work.

Figure 2. Structural assignments of glycoforms in averaged spectra for Asn448 (T45 peptide), within the range of 18.5-22.0 min. The averaged spectrum, containing glycoforms at 2+, 3+, and 4+ charge states was deconvoluted with the Xtract function of the Qual Browser software to produce a “zero” charge state data.

glycopeptides containing the above three glycosylation sitessone peptide for each site with exception of the Asn117-containing peptide for which an additional glycopeptide due to a chymotrypsin-like activity of the digestion enzyme was reported.9 N-Linked glycopeptides found in rt-PA are summarized in Table 1, including a novel glycopeptide identified in this work as will be discussed later in the text. Identification of the glycoforms of each of the glycopeptides was performed by averaging the corresponding spectra in their elution ranges, as identified in the first scan event. This is shown in Figure 2, using the example of T45 peptide from Table 1. Spectra for the range of 18.5-22.0 min were averaged and deconvoluted to generate a “zero” charge state spectrum. Deconvolution partially compensates for differences in ionization efficiencies of various glycoforms and instrument m/z sensitivity dependence (instrument function). Assignment of putative glycan structures to the experimental masses is facilitated by the high mass accuracy of measurements attainable with an LTQ-Orbitrap mass spectrometer and detailed prior knowledge.8 Average mass

error of the assigned masses for the T45 glycoforms relative to theoretical is about 1.2 ppm. Relative amounts of T45 glycoforms, determined from the spectrum in Figure 2, were compared with the amounts calculated from the corresponding RIC peak areas of triply charged ions of each individual glycoform (Table 2). Average relative percentage error between the two methods is about 14%. Furthermore, we noted that this error increases roughly linearly with the molecular weight of an oligosaccharide. At the pH of a reversed-phase separation, sialic acid (pKa 2.2-3.0)20 is expected to be only partially protonated, thus, potentially contributing to a lower ionization efficiency of glycoforms decorated with sialic acids. However, since the masses of T45 complex glycoforms depend not only on the number of capping sialic acids, but on the degree of branching as well, the above result suggests that presence of sialic acids alone cannot account for the observed discrepancies. For most (20) Schauer, R.; Kamerling, J. P. Chemistry, Biochemistry and Biology of Sialic Acids. In Glycoproteins II; Montreuil, J., Vliegenthart, J. F. G., Schachter, H., Eds.; Elsevier: Amsterdam, The Netherlands, 1997; pp 243-372.


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Table 2. Proposed Glycan Structures Attached to Asn448, Based on LC-MS Analysis of rt-PAa

a

Relative amounts are based on RICs for triply charged ion of each of the glycan.

mass spectrometers, sensitivity is a function of m/z and is usually set during tuning. Since m/z values of various triply charged T45 glycoforms vary in a range of 845-1600 amu, an instrument’s nonlinear sensitivity in that region could play a 9748


significant role in determining the relative response of various glycoforms. Recently, Sinha et al.21 reported that during N-glycopeptide mapping with electrospray ionization time-offlight (ESI-TOF) MS, RIC peak area was influenced by the

Figure 3. Reconstructed ion chromatograms (RICs) for triply charged ions of nonglycosylated T45 peptide (a) and for its glycoforms, listed in Table 2 and separated by the degree of branching into biantennary (b), triantennary (c), and tetra-antennary (d).

glycan size and was not as effective for absolute quantification of N-glycosylation in recombinant monoclonal antibody as compared to the results of an N-glycan release assay. The relative percentage error between the two assays, deduced from their data, increased with the size of the glycan. However, for proteins with multiple N-glycosylation sites, such as rt-PA, the N-glycan release assay is significantly more labor intensive, compared to molecules with relatively simple glycosylation profiles such as recombinant monoclonal antibodies investigated in the above study, and requires prior separation and isolation of glycosylation sites. Despite the above disadvantages, LC-MS peptide mapping holds its value for a rapid Nglycosylation profiling, semiquantitative speciation of glycoforms, and identification of glycosylation sites (see the Supporting Information for the details on initial assessment of LC-MS applicability to sample comparability studies). Since the presence of glycans makes glycopeptides more hydrophilic, glycopeptides typically elute in front of a “parent” nonglycosylated peptide (Figure 3). Beyond that, the degree of glycan branching determines the elution order of various glycoforms where peptides modified with more highly branched glycans elute in front of structures with fewer branches. As shown in Figure 3, T45 glycoforms elute in the order of tetra- < tri- < biantennary complex structures. Relative amounts of bi-, tri-, and tetra-antennary structures determined from the RIC peak areas

in Figure 3 are 56%, 34%, and 10%, respectively, and are in reasonably good agreement with previously reported Asn448 glycosylation data of 62%, 30%, and 8%, respectively.8 Identification and Structural Assignments of the Novel N-Glycosylation Site at Position 142. An examination of elution profiles of glycopeptides (Figure 1) indicated the presence of a potentially novel site, eluting between 27.5 and 29.0 min as shown in detail in Figure 4a. For the initial assessment, average spectra for this time range were examined for the presence of glycopeptides (Figure 5). A series of peaks with mass differences of 146, 162, 203, and 291 Da clearly indicated the presence of oligosaccharides in this region. The observed masses, however, did not match any of the masses of known tryptic glycopeptides from rt-PA, even when missed cleavages and variations in enzyme specificity were considered. Thus, the likelihood of a potentially novel glycosylation site was raised. Since the N-glycan release experiments were not performed in this study, the possibility of either N- or O-linked glycosylation was considered. Masses of all rt-PA in-silico digested tryptic peptides with potential glycosylation sites at Asn (for N-linked) and Ser and Thr (for O-linked) were modified by masses of the most common oligosaccharides experimentally observed in CHO expressed therapeutic glycoproteins. By searching the experimental deconvoluted masses Analytical Chemistry, Vol. 81, No. 23, December 1, 2009

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Figure 4. Identification and elution profiles for the N-glycosylated T13 (a-c) and O-fucosylated T8 (d-f) peptides: reconstructed ion chromatograms (RICs) for Hex, Hex-HexNAc, and NANA oxonium ions (a-d) (at “in-source” CID conditions), nonglycosylated T13 (b), and fucosylated T8 (e) peptides, and their major glycoforms (c and f).

(Figure 5) detected in the range of 27.5-29.0 min against the mass list of potential glycopeptides, we observed an excellent match between the experimental data and the masses of a number of T13 (136-145) peptide glycoforms. This peptide (Table 1) has two Asn residues, with one Asn in position 142 being part of an Asn-Tyr-Cys sequence triplet. As will be discussed later, Asn from a general Asn-Xaa-Cys motif can be N-glycosylated. To further confirm our findings, an online glycopeptide prediction program, GlycoMod (http://www. expasy.org/tools/glycomod, Swiss Institute of Bioinformatics), was applied to identify the possible oligosaccharide structures observed on rt-PA from the list of experimentally determined masses (Figure 5). The process further confirmed T13 peptide glycosylation. Reliability of the identification was facilitated by the accurate mass measurements attainable by LTQ-Orbitrap MS. As summarized in Table 3, experimental masses are on average within 0.5 ppm of the masses for the proposed glycan structures. For detailed structural characterization of glycans, multiple analytical methods such as methylation analysis, 1H NMR spectroscopy, and anion-exchange separation of enzymatically released oligosaccharides are typically needed and were previously used in rt-PA glycosylation studies.8 In this work, 9750


however, we have proposed oligosaccharide structures by matching their accurate masses to the commonly occurring mammalian oligosaccharide structures. In addition, CID MS2 spectra for the major glycopeptides eluting in the range of 27-29 min were evaluated to confirm the structural assignment for the glycans (see the Supporting Information, Figure S-1). Our data suggest that the major oligosaccharides modifying Asn142 are biantennary monosialo and asialo hybrid and Man5 type structures. Elution profiles of the proposed structures for major T13 glycans are shown in Figure 4c. These glycoforms elute in front of the T13 nonglycopeptide (Figure 4b), which further supports identification of T13 glycosylation. Relative amounts of the proposed structures were estimated from RIC peak areas and summarized in Table 3. Our data suggest that the total Nglycosylation of the T13 peptide is at about 1%. These low levels of glycosylation could easily explain the fact that this site was not detected in previous studies of rt-PA glycosylation performed over a decade ago.7-12 Improved capabilities and sensitivity of modern analytical technology undoubtedly facilitates detection of low-level modifications. (21) Sinha, S.; Pipes, G.; Topp, E. M.; Bondarenko, P. V.; Treuheit, M. J.; Gadgil, H. S. J. Am. Soc. Mass Spectrom. 2008, 19, 1643–1654.

Figure 5. Proposed structural assignments for a novel glycosylation site at Asn142 (T13 peptide) in averaged spectra, within the range of 27.5-29.0 min. The averaged spectrum, containing glycoforms at 2+ and 3+ charge states, was deconvoluted with the Xtract function of the Qual Browser software to produce a “zero” charge state data. Table 3. Proposed Glycan Structures at a Novel Asn142 N-Glycosylation Site Identified during LC-MS Analysis of rt-PAa

a

Relative amounts are based on RICs for doubly charged ion of each of the glycan.


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It is a general understanding that the consensus sequence, Asn-Xaa-Ser/Thr, where Xaa is any amino acid except Pro, is prerequisite to oligosaccharyltransferase recognition. Furthermore, local folding around the potential N-glycosylation site determines the efficiency of glycosylation. In the early 1980s it was shown, however, that the sequence motif Asn-Xaa-Cys from a model hexapeptide can serve as a substrate to the N-glycosyltransferases.22 This result indicated that, in addition to an AsnXaa-hydroxy motif, the Asn-Xaa-Cys triplet is also capable of fulfilling conformational and catalytic prerequisites for the site to be recognized and glycosylated by corresponding N-glycosyltransferases. This suggests that, in vivo, glycosylation should, in principle, be possible. This assumption was supported by a comparison of the standardized transfer rates of the Asn-Gly-Cys and Asn-Gly-Ser sequons in hexapeptides, which only differ by a factor of 2-3.22 This result indicated that the N-glycosylation at Asn-Xaa-Cys sequon should not be rare. However, to date only a limited number of proteins have been identified to be glycosylated at that site, as summarized by Sato et al.23 (see the Supporting Information for the detailed list of references), with more recent examples being human transferrin24 and crystatin F.25 Reported ranges of N-glycosylation for the Asn-Xaa-Cys sequon range from 1% to 100%. For example, Giuffrida et al.26 showed that only 1% of R-lactalbumin from human milk is N-glycosylated at the Asn-IleCys motif. Researchers suggested that the low levels of glycosylation may reflect a kinetic competition between glycosylation and the rate of protein synthesis or folding. According to the Bause and Legler postulate, a hydrogen-bond donor function in the side chain of the hydroxy or thiol group of the amino acid of the motif sequence is required for glycosyl transfer.27 Formation of a disulfide bridge impeding the participation of Cys in the glycosylation reaction could be a sequent of this mechanism. Miletich and Broze28 postulated that glycosylation at the AsnXaa-Cys sequence in protein C is intrinsically less favorable than at the Asn-Xaa-Ser/Thr site and is therefore the most likely site skipped when the protein is being made rapidly. As a consequence of the hydrogen bond acceptor theory, Asn142 should lose its potential glycosylation site status after the disulfide bond involving the Cys144 has formed. In a properly assembled rtPA, Cys144 is bonded to Cys168 by a disulfide. According to the authors’ postulate, the length of time that Asn142 is in the lumen of the endoplasmic reticulum (ER), where cotranslational protein glycosylation occurs, and is available for glycosylation before protein folding and disulfide formation involving Cys144 occurs, is longer when protein is made at a slower rate. Although it is outside the scope of this work, it would be interesting to determine how the rate of rt-PA production affects Asn142 glycosylation. Also, it can be speculated that Asn142 glycosylation can impede proper rt-PA folding and oxidation (22) Bause, E.; Legler, G. Biochem. J. 1981, 195, 639–644. (23) Sato, C.; Kim, J. H.; Abe, Y.; Saito, K.; Yokoyama, S.; Kohda, D. J. Biochem. 2000, 127, 65–72. (24) Satomi, Y.; Shimonishi, Y.; Takao, T. FEBS Lett. 2004, 576, 51–56. (25) Colbert, J. D.; Plechanovova´, A.; Watts, C. Traffic 2009, 10, 425–437. (26) Giuffrida, M. G.; Cavaletto, M.; Giunta, C.; Neuteboom, B.; Cantisani, A.; Napolitano, L.; Calderone, V.; Godovac-Zimmermann, J.; Conti, A. J. Protein Chem. 1997, 16, 747–753. (27) Kasturi, L.; Eshleman, J. R.; Wunner, W. H.; Shakin-Eshleman, S. H. J. Biol. Chem. 1995, 270, 14756–14761. (28) Miletich, J. P.; Broze, G. J., Jr. J. Biol. Chem. 1990, 265, 11397–11404.

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of Cys144 to a disulfide. To date there is no information available on the presence of low levels of reduced Cys144 in rt-PA. Characterization of the Novel Asn142 N-Glycosylation Site by ETD Fragmentation. ETD is a recently developed, nonergodic fragmentation technique which has been proven to preserve labile modifications, such as glycosidic bonds, while fragmenting the peptides’ backbone to generate c and z product ions.29-32 To elucidate the exact site of glycan attachment within the amino acid sequence of the T13 peptide, we applied this technology during online LC-MS. Initially, doubly charged precursors of the T13 peptide, modified with the top five most abundant glycan structures, as listed in Table 3, were selected for ETD MS2 experiments. Despite the relatively lower abundance of the T13 glycopeptide decorated by the Man5 structure, it produced the best quality ETD MS2 data compared to other four glycoforms. Man5 has the lowest mass among the top five glycoforms in Table 3. It is not quite clear at present whether and how mass (size) of a glycan and presence of acidic saccharides, such as sialic acid, affect ETD efficiency. The T13 glycopeptide with Man5 was selected for further studies. It is well-known that ETD efficiency improves as the peptides’ charge state increases. Although the signal intensity of a triply charged T13 Man5-peptide was 2-fold less compared to a doubly charged precursor with only 3300 counts at the top of the elution profile, this charge state was included in the investigation. ETD fragmentation of triply and doubly charged parent precursors provided complementary information, facilitating glycosylation site identification, as shown in Figure 6. ETD fragmentation pattern obtained for the doubly charged T13 Man5-peptide (Figure 6b) resembles the ETD spectrum of the unmodified T13 peptide (see the Supporting Information, Figure S-2). Even without a priori considerations of a likely glycosylation motif, ETD fragmentation allowed unambiguous identification of the glycosylation site. The occurrence of unmodified c5 and c6 fragment ions in the ETD MS2 spectrum of the triply charged precursor indicates that residues 136-141 do not contain the modification. In contrast, ETD MS2 spectrum of the doubly charged precursor exhibited abundant z4-z9, c7, and c9 ions shifted by 1216.4 Da, which is the mass of the Man5 structure. The mass shifts of fragment ions indicated that glycosylation of T13 peptide is at position 142. This result further confirmed our conclusions based on general considerations of N-glycosylation of the Asn-Xaa-Cys triplet. Detection of Fucose-O-Thr61 Elongation. Peptide T8, modified with O-linked fucose at Thr61, elutes at 68.3 min (Figure 4e), which is approximately 1.6 min earlier than nonglycosylated T8. Careful examination of RICs for characteristic ions in this region (Figure 1) showed a weak but distinct signal from HexNAc and Hex-HexNAc oxonium ions in front of O-fucosylated T8, as shown in detail in Figure 4d. The presence of additional saccharides on this peptide would be expected to result in shorter retention times for these new glycoforms. MS spectra in this time (29) Wuhrer, M.; Catalina, M. I.; Deelder, A. M.; Hokke, C. H. J. Chromatogr., B 2007, 849, 115–128. (30) Mikesh, L. M.; Ueberheide, B.; Chi, A.; Coon, J. J.; Syka, J. E. P.; Shabanowitz, J.; Hunt, D. F. Biochim. Biophys. Acta 2006, 1764, 1811– 1822. (31) Wu, S. L.; Hu ¨ hmer, A. F.; Hao, Z.; Karger, B. L. J. Proteome Res. 2007, 6, 4230–4244. (32) Good, D. M.; Wirtala, M.; McAlister, G. C.; Coon, J. J. Mol. Cell. Proteomics 2007, 6, 1942–1951.

Figure 6. Identification of the novel N-glycosylation attachment site of the T13 (LGLGNHNYCR) peptide with Man5 glycan structure using ETD fragmentation of a 3+, m/z 807.7 (a) and a 2+, m/z 1211.0 (b) parent ions. The asterisk indicates the presence of Man5 glycan on a peptide or its fragment. Fragment ions marked with a # are due to the losses of side chains during ETD.

range were averaged and evaluated for the presence of additional glycoforms. Accurate mass measurements revealed the presence of glycoforms with masses shifted by 162.1, 203.1, 365.1, and 656.2 Da relative to the mass of O-fucosylated T8 peptide. Furthermore, in a separate experiment, CID MS2 spectra for triply charged precursor ions of the novel T8 glycoforms exhibited neutral losses, characteristic to CID of glycopeptides, and partial backbone fragmentation consistent with the sequence of the T8 peptide itself (see the Supporting Information, Figure S-3). This observation is consistent with consideration of O-fucose elongation with HexNAc, (Hex)(HexNAc), and (NANA)(Hex)(HexNAc) to bi-, tri-, and tetrasaccharides. Elution profiles of these glycoforms are shown in Figure 4f. O-Fucose elongation has been reported for several protein contexts, including EGF-like and thrombospondin type 1 (TSR) repeats.15,33 As several glycoproteins with an EGF-like domain, including human factor IX34 and Notch1 protein,35 have been identified to carry O-fucose elongated with the NANA-Gal-GlcNAc-Fuc-O-Ser tetrasaccharide, so it is not unreasonable to expect occurrence of elongation in rt-PA. We determined that approximately 0.7% of O-fucosylated Thr61 is elongated. General Considerations. In general, carbohydrates play a significant role in various biological functions of proteins, including modulation of interaction with receptors, protein folding, stabilization, regulating activity, binding, and pharmacokinetic properties

of proteins they modify. Walsh and Jefferis36 pointed out that effect(s) of a post-translational modification (PTM) on a biopharmaceutical must be evaluated from the point of view of its functional and safety consequences, which can be determined through clinical evaluation, rather than from the structure of the PTM alone. In case of rt-PA, depending on the occupancy at Asn184, type I and type II variants show differences in the fibrin-dependent catalytic activity and rate of plasma clearance.37,38 It is therefore important to monitor the manufacturing consistency of Asn184 glycosylation for judging product quality. On the other hand, there are multiple examples of different products which, although they have different glycosylation profiles, are still safe and effective and differences in glycosylation alone do not automatically have a negative or positive effect on products’ biotherapeutic properties.36 Therefore, the general conclusion has to be that, irrespective of the level, modification by itself has no significance since its importance depends solely on the interaction it has with a biological system. Needless to say, one has to be able to detect the PTMs in the first place. In this case, the low levels of Asn142 N-glycosylation and fucose-O-Thr61 elongation make it difficult to determine the biotherapeutical significance of these modifications in rt-PA. However, their very low abundance would suggest that they have little if any impact on the current therapeutic use of rt-PA.

(33) Shao, L.; Hatliwanger, R. S. Cell. Mol. Life Sci. 2003, 60, 241–250. (34) Harris, R. J.; van Halbeek, H.; Glushka, J.; Basa, L. J.; Ling, V. T.; Smith, K. J.; Spellman, M. W. Biochemistry 1993, 32, 6539–6547. (35) Moloney, D. J.; Shair, L. H.; Lu, F. M.; Xia, J.; Locke, R.; Matta, K. L.; Haltiwanger, R. S. J. Biol. Chem. 2000, 275, 9604–9611.

(36) Walsh, G.; Jefferis, R. Nat. Biotechnol. 2006, 24, 1241–1252. (37) Hotchkiss, A.; Refino, C. J.; Leonard, C. K.; O’Connor, J. V.; Crowley, C.; McCabe, J.; Tate, K.; Nakamura, G.; Powers, D.; Levinson, A.; Mohler, M.; Spellman, M. W. Thromb. Haemostasis 1988, 60, 255–261. (38) Baenziger, J. U. J. Clin. Invest. 1994, 93, 459.


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Table 4. rt-PA Glycosylation Summarya speciation site 117

Asn Asn142 Asn184 Asn448 Thr61 a

occupancy, %

type

major form(s)

99.6 0.9 56.1 99.9 98.4

high mannose complex/hybrid complex/hybrid complex/hybrid O-linked fucosylation

Man5:Man6:Man7 biantennary bi:tri:tetra bi:tri:tetra Fuc:elongated Fuc

this study 63:32:5 55:41:4 56:34:10 99.3:0.7

ref 8 50:41:9b ND 57:37:6 62:30:8 ND

Relative amounts in this study are based on RICs for the most abundant ion of each of the glycan. b Reported in ref 16.

In our study we took a retrospective look at a number of rt-PA samples manufactured at different times throughout the rt-PA commercial production. In all the cases, N-glycosylation of the T13 peptide and the fucose-O-T8 elongation were detected in the range of 0.8-1.3% and 0.7-0.8%, respectively, indicating that these PTMs are not new in the sense of historic rt-PA production and that their identification was enabled solely by recent advances in analytical technology. Findings reported in this study may be of interest to glycobiologists and our general understanding of glycosylation mechanisms, rather than consequences they have on rt-PA product quality. State-of-the-art mass spectrometry with a combination of high sensitivity, accuracy, and dynamic range provides a new ability to discover and characterize low-level protein variants and PTMs. For example, Yu et al.39 recently reported a detection of low levels of codon-specific misincorporation of asparagine residues at multiple serine positions of a monoclonal antibody sequence ranging from 0.01% to 0.2%, consistent with published predictions for in vivo translation error rates. The authors concluded that recent advances in the state-of-the-art analytical technology can now probe at levels as at the levels close to the limit of the cellular ability to ensure amino acid sequence fidelity. Such an ability to detect unknowns, in general, is an important criterion by itself as it validates current approaches for analytical characterization. CONCLUSIONS LC-MS methods have improved tremendously since the introduction of rt-PA in the mid 1980s. The original identification of the glycosylation sites in rt-PA was performed by identifying glycopeptides in the tryptic maps using amino acid analysis and Edman degradation,6 fast atom bombardment (FAB) MS analysis of glycopeptides7 and of released and fractionated glycans for their structural characterization.8 Online LC-MS characterization of glycopeptides in rt-PA was first performed after the introduction of ESI,9 which also facilitated identification of O-fucosylation at Thr61.13 Identification of glycopeptides was limited to characterization of the major glycosylation sites with a strong TIC response. Thus, low-level glycopeptides were undetected. (39) Yu, X. C.; Borisov, O.; Alvarez, M.; Michels, D. A.; Wang, Y. J.; Ling, V. Anal. Chem., published online, October 23, 2009.

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In this study, LC-MS was operated in a fast switching mode of “in-source” fragmentation conditions to profile glycans in rtPA. The method relies on the signal from characteristic oxonium ions,17 due to partial fragmentation of oligosaccharide linkages, and is more sensitive compared to the identification based on a simple visual TIC inspection. This method was successful in identifying the elution regions of major previously reported tryptic glycopeptides. Accurate mass measurements facilitate assignments of putative oligosaccharide structures. The relative amounts of glycans determined in this work are in reasonable agreement with published values as summarized in Table 4. Although this method can hardly be claimed as quantitative, it can be useful for fast glycoprofiling in cases where semiquantitative relative amounts of glycoforms are sought with potential for use in monitoring production consistency and comparability screening. In addition to the previously reported structures, we detected two novel glycopeptides. In one case, a novel N-glycosylation at position 142 was identified to be due to glycosylation of an atypical Asn-Xaa-Cys sequence motif. The glycosylation site was confirmed using ETD as a fragmentation technique. Although Asn142 is only about 1% occupied by predominantly biantennary hybrid structures, it was readily identifiable by current LC-MS methods. In another case, elongation of O-fucose to bi-, tri-, and tetrasaccharides at Thr61 was detected at levels of about 0.7%. Both of these glycoforms are low in abundance and were likely to have been below the detection capabilities of analytical instruments used a decade ago when the original characterization of rt-PA was performed. In conclusion, as analytical technology becomes more sensitive, detection of low-level modifications is becoming routine. ACKNOWLEDGMENT We thank Tomasz Baginski, Ola Saad, and Surinder Kaur of Genentech, Inc. for critical reading of the manuscript. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review July 6, 2009. Accepted October 9, 2009. AC901498K

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