Hong Wang, Xi Chen, Xiaoxi Zhang, Wei Zhang, Yan Li, Hongrui Yin, Hong Shao, and Gang Chen

J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00921 • Publication Date (Web): 26 Jan 2016

 

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Hong Wang†, Xi Chen‡, Xiaoxi Zhang¶, Wei Zhang¶, Yan Li§, Hongrui Yin†, Hong Shao†,

Gang Chen*†

† Shanghai Institute for Food and Drug Control, Shanghai, 201203, China

‡ Waters Corporation, Shanghai, 201206, China

¶ Thermo Fisher Scientific, Shanghai, 201206, China

§ Shanghai Techwell Biopharmaceutical Corporation, Shanghai, 201108, China

* Corresponding author: Tel.:+86-21-50798175; fax: +86-21-50798176.

E-mail address: chengang@smda.gov.cn

 

Glycosylation is an important PTM and is critical for manufacture and efficacy of therapeutic glycoproteins. Glycan significantly influences the biological properties of human follicle-stimulating hormone (hFSH). Using a glycoproteomic strategy, this study compared the glycosylation of a putative highly purified FSH (uhFSH) obtained from human urine with that of a recombinant human FSH (rhFSH) obtained from Chinese hamster ovary (CHO) cells. Intact and subunit masses, N-glycans, N-glycosylation sites, and intact N- and O-glycopeptides were analyzed and compared by mass spectrometry. Classic and complementary analytical methods, including SDS-PAGE, isoelectric focusing, and the Steelman-Pohley bioassay were also employed to compare their intact molecular weights, charge variants, and specific activities. Results showed that highly sialylated, branched, and macro-heterogeneity glycans are predominant in the uhFSHcompared with rhFSH. The O-glycopeptides of both hFSHs, which have not been described previously, were characterized herein. A high degree of heterogeneity was observed in the N-glycopeptides of both hFSHs. The differences in glycosylation provide useful information in elucidating and in further investigation of the critical glycan structures of hFSH.

 

Key words: follicle-stimulating hormone, uhFSH, rhFSH, glycosylation, mass spectrometry

 

Human follicle-stimulating hormone (hFSH) plays a key role in the development and function of the reproductive system and is used clinically to stimulate follicular maturation for in vitro fertilization and treatment of an ovulatory women.1 hFSH is a heterodimeric structure consisting of non-covalently linked α- and β-subunits. Each subunit contains two N-glycosylation sites carrying sialylated complex type N-glycans.2 The oligosaccharide composition, branching pattern, and number of sialic acid residues of hFSH are markedly variable giving rise to multiple glycoforms of hFSH. Two classes of hFSH-containing pharmaceutical preparations currently exist; those derived from the urine of post-menopausal women (uhFSH) and those manufactured using recombinant DNA technology (rhFSH). These preparations possess identical amino acid sequence,although their terminal sialylation, pI, half-life, and effect in clinical application vary.3 Comparative clinical studies have revealed the differences in oocyte quality and clinical outcome between rhFSH and uhFSH.4

 

The oligosaccharide moiety is critical in determining the pharmacological properties, including stability, solubility/bioavailability, in vivo activity, pharmacokinetics, and immunogenicity of therapeutic glycoproteins.5 The two major types of oligosaccharide attached to therapeutic glycoproteins are the N-linked and O-linked glycans. Glycans significantly influence the biological properties of hFSH. Glycans attached to the α-subunit are critical for dimer assembly, integrity, secretion, and signal transduction, whereas β-subunit glycans are important for dimer assembly and secretion.6 The sialylation and complexity of hFSH oligosaccharides affect endocrine activity and expression of genes regulating granulosa cell function.7,8 The classic Steelman–Pohley bioassay in rats showed that FSH exhibiting high level of sialylation possesses long half-life in vivo biopotency.9 Moreover, the threshold of ovarian follicles and the dynamics of follicular growth are influenced by FSH glycosylation.10 Some studies have revealed that uhFSH has longer half-life than rhFSH because it contains more sialic acids.11 Considering the importance of hFSH glycosylation to the biological activity of hFSH, evaluating the difference in glycosylation in rhFSH and uhFSH intended for clinical use is essential.

 

However, analysis of protein glycosylation is a challenging task because of the variable glycoside linkages, branching, and numerous isomers in hFSH. Liquid chromatography-mass spectrometry (LC-MS) has become an invaluable combination of technologies for detection, quantification, comparison, and further elucidation of glycan structure. Alternative fragmentation technologies, including higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), can extensively characterize not only N-glycopeptides but also O-glycopeptides.12 Moreover, several studies have already mapped hFSH glycosylation. The oligosaccharide composition of commercial FSH preparations was evaluated using RP-HPLC/IT-TOF MS.13 The results showed the highly sialylated and branched glycans in uhFSH compared with rhFSH expressed in rodent cell lines. Site-specific analysis of all four glycosylation sites in rhFSH was also accomplished using Q-TOF MS.1 In addition, the glycan moiety of rhFSH produced in CHO cells was analyzed using a combination of LC and MS techniques, including both matrix-assisted laser desorption ionization (MALDI) and electro-spray ionization (ESI) MS.14 Quantitation of the level of sialylation and of antennarity of N-glycans was obtained using glycan mapping methods. FSH glycosylation micro-heterogeneity in pituitary and urinary hFSH was evaluated using nano-ESI-MS.15

 

Using a glycoproteomic strategy, we compared the glycosylation pattern of uhFSH with that of rhFSH produced in CHO cells. The N-glycan chains labeled with 2-aminobenzamide (2-AB) were analyzed and quantified using hydrophilic interaction chromatography (HILIC)-based LC-MS. The N-glycosylation sites and the site-specific glycan structures were also revealed. The novel HCD product-dependent ETD (HCD-pd-ETD) workflow was used in revealing the O-glycosites and site-specific O-glycans. In addition, the charge variants and sialic acid contents were determined, and in vivo bioassay was performed. Higher level of sialylation, antennary, and macro-heterogeneity were detected in uhFSH than in rhFSH. Moreover, NeuGc residue was found in rhFSH and is possibly immunogenic. Highly heterogeneous N-glycosylation patterns were also observed in both hFSHs. Two O-glycosylation sites were discovered in rhFSH, whereas five O-glycosylation sites were discovered in uhFSH. O-glycosylation in both hFSHs has not yet been described. The differences in glycosylation provide deeper insight into the critical glycosylation structures of hFSH.

 

One lot of PuregonR-HP of 50 IU/0.5mL and two lots of PuregonR-HP of 100 IU/0.5mL (rhFSH) (Organon, Oss, Netherlands) were purchased in China. All strengths were presented as liquor. One lot of high purity uhFSH (>98%) was obtained directly from the manufacturer, Shanghai Techwell Biopharmaceutical Company (Shanghai, China), as active pharmaceutical ingredient. Clean gel IEF was obtained from Amersham Biosciences (Piscataway, NJ). Dithiothreitol (DTT), iodoacetamide (IAM), NH4HCO3, trypsin, 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, SA) were purchased from Sigma-Aldrich (St. Louis, MO). Chymotrypsin was obtained from Roche (Penzberg, Germany). PNGase F, from New England Biolabs (Ipswich, MA); 10kDa MWCO centrifugal filters, from Millipore (Bedford, MA); and porous graphitized carbon (PGC) columns, from Grace (Columbia, MD). Glycoclean column and 2-AB labeling kit were obtained from Prozyme (San Leandro, CA). All other reagents were purchased from Sigma (St. Louis, MO) or Fluka (Bucho, Switzerland).

 

The rhFSH isolation by SEC column was performed as previously described.16 For this study, three lots of PuregonR injection were dispensed and combined, then loaded on to a Sephadex G-25 column (GE Healthcare life science) at a flow rate of 2 mL/min under isocratic condition of buffer A (2 mM phosphate, pH 7.4). The baseline of the flow through was monitored at 215 and 280 nm until a return to baseline was observed. The eluted rhFSH was collected and applied to a 10 kDa MWCO Centrifuge device (50 mL). The centrifuge was spun for 1 h at 3,000 rpm at 4°C and washed with buffer of 5 mM phosphate containing 150 mM NaCl pH 7.4. After concentration, fractions were combined and stored at -80°C. The isolated rhFSH concentration was determined by SEC-HPLC (superdex 75, GE Healthcare life science), and the protein purity, quality and recovery were compared to PuregonR.

 

For intact molecular weight assay, the hFSH samples were subjected to 10% SDS-PAGE under non-reducing condition. Gels were stained by Coomassie R-250. The subunits of hFSH were analyzed in linear mode by the 4800 Plus MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Framingham, MA).14 The reduced hFSH subunits were further analyzed by the Acquity UPLC system connected on-line to a quadrupole time-of-flight tandem mass spectrometer (Xevo G-2S, Waters Corporation, Milford, MA). The column was a Waters Acquity UPLC BEH C4 column (2.1 mm × 100 mm, 1.7 μm particle). The flow rate was 0.2 mL/min using a gradient from 15% to 25% solvent B (100% acetonitrile with 0.1% formic acid) in 25 min at a column temperature of 80°C. Solvent A was 0.1% formic acid in water. For mass spectrometric analysis, a MS data acquisition method was employed with a scan range m/z 500-2500.

 

A commercial dried polyacrylamide gel, Clean gel IEF, was rehydrated in a solution containing an appropriate composition of carrier ampholytes necessary to create the desired pH gradient (3–6). The separated proteins and pI marker were stained by Coomassie blue.

 

The sialic acid contents of both hFSHs were quantitatively estimated using a colorimetric resorcinol-hydrochloric acid method. The method was followed ChP 2010 Vol III: Appendix VI C, 35. The mean relative content and SD were determined for two replicates per sample.

 

For N-glycosylation site identification and O-glycopeptide analysis, both hFSHs were reduced by incubating with 10 mM DTT for 30 min at 57°C and alkylated with 20 mM IAA at room temperature for 1h in the dark. One molar NH4HCO3 was diluted in the sample to make its final concentration to 50 mM. Deglycosylation was performed by adding 300IU PNGase F at 37°C overnight. Trypsin was added at 1:50 w/w and incubated at 37°C for 14 h. Digestion was quenched by adding 10% TFA.For N-glycopeptide analysis, samples of each hFSH were reduced and alkylated.Chymotrypsin was added at 1:25 w/w in 100 mM Tris-HCl, 10mM CaCl2 (pH 7.8).Enzymatic digestion was performed overnight at 25°C.

 

The oligosaccharides of hFSH were released by PNGase F and desalted on a non-porous graphitized carbon. Then 2-AB labeling and purification of N-glycans were performed as described in previous study.17

 

Analysis of 2-AB labeled N-glycans was performed on a Waters Acquity UPLC BEH glycan column (2.1 mm × 150 mm, 1.7 μm particle) using an Acquity UPLC with a fluorescence detector (Waters Corporation, Milford, MA).18 The glycans were analyzed using mobile phase A, 50 mM ammonium formate (pH 4.4), and mobile phase B,acetonitrile. The gradient was 70-53% B in 35 min with a flow rate of 0.4 mL/min and

column temperature of 40°C. Samples were injected in 80% acetonitrile. The fluorescence detection was carried out using an excitation wavelength of 330 nm and an emission wavelength of 420 nm. The eluted positions of the N-glycans were determined in glucose units (GU) by comparison with a standard dextran hydrolyzate 2-AB labeled (dextran ladder).19 The LC-MS data were acquired on Xevo G-2S (Waters Corporation, Milford, MA). The interested parent ions were selected for MS2 data acquisition.20

 

A multiplexed data acquisition method (MSE) was employed for mass spectrometric analysis of tryptic digest of hFSHs. The instrument and analyzed method were described previously.20

 

N-Glycopeptides were analyzed by the Acquity UPLC system connected on-line to Xevo G-2S. The column was also a Waters Acquity UPLC BEH glycan column (2.1 mm × 150 mm, 1.7 μm particle). The experiment was performed as described elsewhere.21 The flow rate was 0.2 mL/min using a gradient of 1% to 40% solvent B (100% acetonitrile with 0.1% formic acid) in 63 min, followed by an increase to 90% B in 4 min, and then to 90% B in 6 min. Solvent A was 0.1% formic acid in water.

 

O-Glycopeptides were analyzed by nano LC-MS/MS on an Orbitrap Fusion Tribrid MS (Thermo Fisher Scientific, San Jose, CA) coupled to an EASY-nLC System (Thermo Fisher Scientific, San Jose, CA). Peptide mixtures were loaded onto a Magic C18 spray tip 15 cm × 75 μm i.d. column (Michrom Bioresources) and separated at a flow rate of 350 nL/min using a gradient of 8% to 22% solvent B (100% acetonitrile with 0.1% formic acid) in 54 min, followed by an increase to 35% B in 15 min, and then to 90% B in 10 min and held for another 6 min. Solvent A was 0.1% formic acid in water.Data acquisition was performed under data dependent acquisition (DDA) with HCD-pd-ETD. The parameters settings were: top speed mode with 3 s cycle time; FT MS: scan range (m/z) = 350−2000; MS resolution = 120K; MS2 resolution=30K; other parameters followed a previous report.22 To restrict the ETD MS2 data acquisition to true O-glycopeptide precursors, the preceding HCD can be applied in producing diagnostic glyco-oxonium ions (138.0545, 204.0867 and 366.1396) using as a filtering criterion to trigger ETD in the HCD-pd-ETD mode.

 

N-glycan data were processed using UNIFI 1.7 with Glycobase 3+ (Waters Corporation, Milford, MA) for N-glycan structure. The peak area of chromatography was calculated for relative glycan quantification. The mean relative content and SD were determined for three replicates per sample. The MS2 data analysis was performed with MassLynx 4.1 data system. The data of intact hFSH subunits were deconvoluted and analyzed by UNIFI 1.7. The observed N-glycans structures were set as amino acid modification for deconvolution (mass error <50 ppm).

 

The LC-MSE data were processed using Biopharma Lynx 1.3.3 (Waters Corporation, Milford, MA) for N-glycosylation sites analysis. The mass tolerance was set at 5 ppm for precursor and 5 ppm for fragment ions, respectively. The identified peptides were confirmed by MSE spectra with at least five b/y fragment ions. The N-glycopeptide data were analyzed by ProteinLynx Global SERVER 3.0 (Waters Corporation, Milford, MA). In order to identify N-glycopeptides in rhFSH and uhFSH digest, N-linked glycosylation was selected as variable modification.

 

For O-glycopeptide identification, the data of HCD-pd-ETD were searched separately using Byonic 2.5.6 (Protein Metrics, San Carlos, CA) with the following search parameters: peptide tolerance = 10 ppm; fragment tolerance = 0.02 Da for both HCD and ETD; missed cleavages = 1; modifications: carbamidomethyl cysteine (fixed), methionine oxidation (common2). Mucin type O-glycan including Hex (mass of 162.0528), HexNAc (mass of 203.0794), HexNAc2 (mass of 406.1587), HexNAc2Hex1 (mass of 568.2116),NeuAc1HexNAc1Hex1 (mass of 656.2276) and NeuAc2HexNAc1Hex1 (mass of 947.3230) were set as database in the Byonic data searching. Spectrum-level FDR was set as auto cut. The PSM with score ≥100 and mass error <10 ppm were accepted.

 

Protein contents were assessed by SEC-HPLC using BSA as standard. The in vivo bioactivity of hFSH was assessed according to current US and European Pharmacopoeias by the traditional Steelman-Pohley human chorionic gonadotrophin (hCG) augmentation assay, which measures ovarian hypertrophy following administration of exogenous FSH (in combination with hCG) to immature female rats.23

 

SEC-HPLC results showed that rhFSH, which was efficiently isolated using PuregonR,maintained its overall structural integrity and purity. For more accurate structural comparison between rhFSH and uhFSH, we employed a glycoproteomic strategy in characterizing glycosylation, including determination of intact masses, sialic acid contents, N-glycosylation sites, N-glycan structures, site-specific N- and O-glycans, and relative glycan quantities and specific activities (Figure 1a).

 

The intact rhFSH and uhFSH were analyzed using SDS-PAGE. The bands showed these intact proteins comprising glycosylated α- and β-subunits exhibited similar molecular weights of approximately 43 kDa (Figure 1b). The mass of each subunit was determined using high-resolution MALDI-TOF/TOF mass spectrometer (Figure 1b). The α- and β-subunits have the oretical molecular weights of 10 and 12 kDa, respectively. The mass range of the two subunits of rhFSH was 14–15 kDa, whereas that of the two subunits of uhFSH was 14–18 kDa. The mass of each subunit was further analyzed by UPLC-ESI-QTOF MS (Supporting Information Figure 1). The theoretical molecular masses of subunits and glycan chains were searched in the deconvoluted data. The results showed that the molecular masses of rhFSH subunits were in the range 13,710-17,264 Da, whereas those of uhFSH subunits were at 13,453-17,336 Da (Supporting Information Table 1). These results indicated that both proteins contain heterogeneous glycosylated isoforms. Moreover, the broader mass range of uhFSH suggested that glycosylation in uhFSH is more complex than in rhFSH.

 

Isoelectric focusing (IEF) was used in isolating the hFSH charge analogues mainly according to their sialylation level, which increases the acidity of the isoform. The pI range of the isoform profile of rhFSH was 4–5, whereas that of uhFSH was 4–4.6 (Figure 1c). Therefore, the difference between the isoform profile of rhFSH and uhFSH indicated higher sialylation level in uhFSH.

 

Sialic acid content in each hFSH was further assessed. The sialic acid content of rhFSH was 11.5 ± 0.04 mol/mol protein, whereas that of uhFSH was 13.7 ± 0.04 mol/mol protein (Figure 1d). Sialylation level was higher in uhFSH than in rhFSH, and the uhFSH/rhFSH ratio was 1.19 ± 0.004 (p=0.0004). The sialic acid in hFSHs is highly important in their receptor binding ability, biological activity, and clearance from maternal circulation.24 Thus, the differences in the sialylation level of the hFSHs results in the differences in their biological properties.

 

The N-glycosylation site generally contains an N-X-S/T sequence motif (where X ≠ Pro). The primary sequence of hFSH encodes two N-glycosylation sequons for each subunit;these N-glycosylation sites are located at N52 and N78 of the α-subunit and at N7 and N24 of the β-subunit. In high-resolution MS2 analysis, the conversion of N into D with an increase of 0.98 Da after removing N-glycans using PNGase F can be used in identifying N-glycosylation sites. All of four N-glycosylation sites were identified in hFSHs. Most of the sites were fully occupied by N-glycans, except βN24 in uhFSH, as the partial conversion was observed in mass spectra after deglycosylation (Figure 1e). Partial N-glycosylation of the FSH β-subunit was first reported in recombination bovine FSH.25 Both of the β-subunit carbohydrate residues were demonstrated to determine the metabolic clearance rate and in vivo potency of hFSH.26 The lack of oligosaccharides in the β-subunit possibly results in differences in delivery rates to target tissues and in elimination rates via filtration in the kidney.25

 

The N-linked glycans, released from each hFSH, were labeled with 2-AB at their reducing ends. The chromatography peak area was calculated for relative glycan quantification. The system was calibrated using an external standard of dextran ladder from which the retention time for individual glycan was converted into GU.27 The glycans were analyzed on the basis of their GUs, which were then compared with reference values in the “Glycobase” database for preliminary structural assignment. The preliminary structures of the glycans were further confirmed by their masses detected in MS.

 

Supporting Information Table 2 and Figure 2 show the profiling and relative contents of N-glycans through HILIC separation and fluorescence detection. The results showed that the two hFSHs possessed complexity in their carbohydrates, such as (a) degree of complexity in branching; (b) mainly complex type oligosaccharides; and (c) variations in core-/antenna-fucose and terminal sialic acid residues. However, the mono- and di-sialylated biantennary glycans were predominant in rhFSH at a relative amount of 46.23%, whereas the di- and tri-sialylated species, with bi- or tri-antennary glycans, were the most common forms in uhFSH. Moreover, the bisecting GlcNAc moieties linked to the core mannose residue were present in uhFSH. rhFSH also exhibited a more fucosylated distribution compared with uhFSH. The disialylated biantennary glycan NeuAc2HexNAc4Hex5 showed the highest relative content in both hFSHs despite the difference in sialic acid linkage of the two hFSHs. Approximately 10.20% of the oligosaccharides of rhFSH were not sialylated, whereas all of the glycan chains in uhFSH showed sialylation.

 

Some interesting N-glycans were further analyzed using tandem MS. LC-MS/MS analysis revealed the presence of NeuGc-containing, antenna-fucosylated, and sulfated glycans in rhFSH. The MS2 fragment ions obtained from the 2-AB labeled N-glycan at m/z 1180.43 described a complex type biantennary structure containing a NeuGc residue (Figure 3a). The protonated B1α and B3α ions at m/z 308.1 and 673.2, respectively, suggested the presence of NeuGc residue. Sulfated glycans at m/z 1214.44 were also

identified (Figure 3b). Moreover, the NeuAc residue was found to be sulfated at m/z 376.1. The dehydrated sodiated ions at m/z 538.1 and 741.2 further suggested the occurrence of sulfation.

 

The N-glycans of uhFSH were also investigated and data showed the presence of bisecting core-/antenna-fucosylated isomers. Figure 3c and 3d show the MS2 spectrum of Fuc2NeuAc1HexNAc5Hex5, [M+2H]2+. The two isomers were successfully revealed based on the differences in retention times shown by HILIC. The diagnostic ions indicated that the first isomer contains bisecting biantennary structure with antenna fucosylation, whereas the second isomer is indicative of a triantennary oligosaccharide with core and

antenna fucosylation.

 

rhFSH contains the NeuGc residue, which is an unusual and immunogenic oligosaccharide.28 Moreover, core and antenna fucosylations were both found in the two hFSHs. Fucose addition is as important as addition of sulfate group or sialic acid because it ensures proper binding to appropriate receptors.29 Furthermore, terminal sialic acids and sulfate groups regulate the biological half-life of hFSH.30 Bisecting GlcNAc also increase liver and spleen uptake of glycoproteins that possess the structural feature described above.31 The glycan isomers of uhFSH have been detected in various peaks in chromatograms, indicating the complexity of N-glycosylation.

 

For intact N-glycopeptide analysis, chymotryptic digests of both hFSHs were subjected under UPLC equipped with a HILIC column designed to retain hydrophilic peptides, such as glycosylated peptides. MS2 spectra containing glycan fragment ions representing markers of specific glycopeptides were applied for database searching and glycan elucidation (Figure 4a). Highly heterogeneous glycosylation patterns were observed for each N-glycosylation site of both hFSHs. Supporting Information Table 3 summarizes the glycan structures found at each glycosylation site. Mono-, bi-, tri-, and tetra-antennary sialylated glycans were present in both subunits. These glycans were predominantly of the complex type, although some hybrid forms could also be observed. A few glycan chains at αN78 and βN24 of rhFSH and at αN52 of uhFSH showed high fucosylation level. Several structural differences were apparent in the glycan of rhFSH and uhFSH. In rhFSH, sulfated glycans were present at αN52, as well as in both sites in the β-subunit, whereas no sulfated glycan was observed in uhFSH. Moreover, the glycans containing

NeuGc were identified at αN78 and in both sites of the β-subunit, whereas this structure was not observed in uhFSH. Penta-antennary oligosaccharides are possibly present at αN52 and βN24 of uhFSH. Figure 4b shows the glycans attached at αN52 of each hFSH. The αN52 of rhFSH possessed sulfated complex glycans with and without sialylation,whereas highly fucosylated glycans and hybrid type glycans were observed at the same site in uhFSH.

 

A previous study has reported that the binding between hFSH and G protein-coupled receptors that results in hormonal response is greatly affected by the glycans at particular sites. For instance, the site-directed mutagenesis that occurred at αN52 of hFSH to selectively eliminate the glycan at this position resulted either in increased or in an unaltered receptor binding activity with a reduced receptor activation and signal transduction.32 Therefore, characterization of rhFSH and uhFSH glycopeptides can provide valuable information on their functions.

 

Compared with N-glycosylation, O-glycosylation attached to S or T does not have a consensus motif for potential sites, does not have a common core structure, and are more heterogeneous. To identify O-glycosylation, the hFSH samples were treated with PNGase F to remove the N-glycans and then digested with trypsin. The tryptic peptides were analyzed under DDA with product ion triggered ETD (HCD-pd-ETD). The HCD data are rich in glycan fragments, along with a few peptide cleavage b and y ions, whereas the

complementary ETD preferentially induces cleavages along the peptide backbone providing c and z ions. The most common mucin-type O-linked oligosaccharides were used as a glycan database for data processing.

 

Table 1 shows the identified O-glycopeptides in both hFSHs. Two O-glycosylation sites were assigned in both subunits of rhFSH, whereas five O-glycosylation sites were identified in both subunits of uhFSH. Two glycoforms, namely, HexNAc2Hex1 and HexNAc, were identified in βT6 of rhFSH by both fragmentation modes. Figure 5 shows the b/y and c/z ions of O-glycopeptide NSCELTNITIAIEKEEC#R with a HexNAc2Hex1

residue. Although an N-glycosylation site is also located at the peptide, the presence of b6 and c6 ions confirmed that the HexNAc2Hex1 was attached to βT6. The presence of target glyco-oxonium ion, HexNAc, further confirmed the presence of O-glycans in rhFSH.Only one glycoform, HexNAc2Hex1, was identified in βT6 of uhFSH. The O-glycosylation site, αS43, was also identified in both hFSHs, while αS34, αS39 and βT34 were only assigned in uhFSH. O-glycosylation has not yet been reported in hFSH. Compared with N-glycans, O-glycans are more heterogeneous and exhibit no common core structures;O-glycosylation characterization is thus a great challenge and a systematic approach in analyzing glycosylation is called for. We employed DDA with HCD-pd-ETD on Orbitrap Tribrid MS to obtain reliable information on O-glycosylation. The βT6 of rhFSH and βT6 and βT34 of uhFSH were located close to the N-glycosylation sites. Thus, O-glycosylation, which is accompanied by N-glycosylation, possibly plays a key role in determining the functional properties of hFSH.

 

The oligosaccharides in hFSH determine the half-life of plasma and consequently the in vivo bioactivity of the secreted hormone.6 The in vivo bioactivity of hFSH was further studied herein through the ovarian weight augmentation test. Table 2 shows the protein contents, in vivo bioactivity, and specific activity of rhFSH and uhFSH. The measured in vivo bioactivity of rhFSH corresponded closely with the labeled amount. The specific activity of rhFSH was nearly similar to that of uhFSH. The investigation on the biological effects of hFSH glycosylation has also demonstrated that variations in FSH glycosylation have specific effects on follicular growth, antral formation, and estradiol secretion.7

Therefore, the biological effect of rhFSH and uhFSH must be further investigated.

 

Various strategies were used to map and compare the oligosaccharides of rhFSH and uhFSH. The following were investigated in this study: (a) analysis of intact protein and subunit masses; (b) profiling of N-glycan structures; (c) characterization of site-specific Nand O-glycosylation; (d) determination of N- and O-glycosylation sites; and (e) analysis of charge variations, sialic acid contents, and specific activities. High level of sialylation, antennary, and macro-heterogeneity were observed in uhFSH. Moreover, NeuGc and sulfated glycans were detected in rhFSH. We also elucidated for the first time the

O-glycosylation in both hFSHs. Comparative study on the biological effect of rhFSH and hFSH is further required to reveal the critical structural features of glycans.

 

(1) RP-UPLC-Q-TOF analysis of hFSHs subunits; (2) molecular weights and possible glycan structures of hFSHs subunits; (3) profiling and relative quantification of N-glycans of hFSHs; (4) site-specific characterization of N-glycosylation of rhFSH and uhFSH.

 

This work was supported by Shanghai Sailing Program (15YF1410900), Shanghai Technology Standard Program (14DZ0502100).The authors declare no competing financial interest.

 

(1) Grass, J.; Pabst, M.; Chang, M.; Wozny, M.; Altmann, F. Analysis of recombinant human follicle-stimulating hormone (FSH) by mass spectrometric approaches. Anal.Bioanal. Chem. 2011, 400, 2427–2438.

(2) Green, E. D.; Baenziger, J. U. Asparagine-linked oligosaccharides on lutropin,

follitropin, and thyrotropin. II. Distributions of sulfated and sialylated oligosaccharides on bovine, ovine, and human pituitary glycoprotein hormones. J. Biol. Chem. 1988, 263,36–44.

(3) Lispi, M.; Bassett, R.; Crisci, C.; Mancinelli, M.; Martelli, F.; Ceccarelli, D.; De Bellis, C.;

Mendola, D. Comparative assessment of the consistency and quality of a highly purified FSH extracted from human urine (urofollitropin) and a recombinant human FSH (follitropin α). Reprod. BioMed. Online 2006, 13, 179–193.

(4) Selman, H.; Pacchiarotti, A.; El-Danasouri, I. Ovarian stimulation protocols based on follicle-stimulating hormone glycosylation pattern: impact on oocyte quality and clinical outcome. Fertil. Steril. 2010, 94, 1782–1786.

(5) Li, H. J.; d’Anjou, M. Pharmacological significance of glycosylation in therapeutic. Curr.Opin. Biotechnol. 2009, 20, 678–684.

(6) Ulloa-Aguirre, A.; Timossi, C.; Damian-Matsumura, P.; Dias J. A. Role of glycosylation in function of follicle-stimulating hormone. Endocrine 1999, 11, 205–215.

(7) Loreti, N.; Fresno, C.; Barrera, D.; Andreone, L.; Albarran, S. L.; Fernandez, E, A.;Larrea, F.; Campo, S. The glycan structure in recombinant human FSH affects endocrine activity and global gene expression in human granulosa cells. Mol. Cell. Endocrinol. 2013,366, 68–80.

(8) Loreti, N.; Ambao, V.; Andreone, L.; Trigo, R.; Bussmann, U.; Campo, S. Effect of sialylation and complexity of FSH oligosaccharides on inhibin production by granulosa cells. Reproduction 2013, 145, 127–135.

(9) D’Antonio, M.; Borrelli, F.; Datola, A.; Bucci, R.; Mascia, M.; Polletta, P.; Piscitelli, D.;

Papoian, R. Biological characterization of recombinant human follicle stimulating hormone isoforms. Hum. Reprod. 1999, 14, 1160–1167.

(10) Vitt, U. A.; Kloosterboer, H. J.; Rose, U. M.; Mulders, J.W.; Kiesel, P. S.; Bete, S.;Nayudu, P. L. Isoforms of human recombinant follicle-stimulating hormone: comparison of effects on murine follicle development in vitro. Biol. Reprod. 1998, 59, 854–861.

(11) Barrios-de-Tomasi, J.; Timossi, C.; Merchant, H.; Quintanar, A.; Avalos, J. M.;Andersen, C. Y.; Ulloa-Aguirre, A. Assessment of the in vitro and in vivo biological activities of the human follicle-stimulating isohormones. Mol. Cell. Endocrinol. 2002, 186,189–198.

(12) Marino, K.; Bones, J.; Kattla, J. J.; Rudd, P. M. A systematic approach to protein glycosylation analysis: a path through the maze. Nat. Chem. Biol. 2010, 6, 713–723.

(13) Lombardi, A.; Andreozzi, C.; Pavone, V.; Triglione, V.; Angiolini, L.; Caccia, P.Evaluation of the oligosaccharide composition of commercial follicle stimulating hormone preparations. Electrophoresis 2013, 34, 2394–2406.

(14) Gervais, A.; Hammel, Y. A.; Pelloux, S.; Lepage, P.; Baer, G.; Carte, N.; Sorokine, O.;Strub, J. M.; Koerner, R.; Leize, E.; Van Dorsselaer, A. Glycosylation of human recombinant gonadotrophins: characterization and batch-to-batch consistency.Glycobiology 2003, 13, 179–189.

(15) Bousfield, G. R.; Butnev, V. Y.; Rueda-Santos, M. A.; Brown, A.; Hall, A. S.; Harvey,D. J. Macro- and micro-heterogeneity in pituitary and urinary follicle-stimulating hormone glycosylation. J. Glycomics Lipidomics 2014, 4, 125.

(16) Liu, C.; Dong, S.; Xu, X. J.; Yin, Y.; Shriver, Z.; Capila, I.; Myette J.; Venkataraman G. Assessment of the quality and structural integrity of a complex glycoprotein mixture following extraction from the formulated biopharmaceutical drug product. J. Pharm.Biomed. Anal. 2011, 54, 27–36.

(17) Nuck, R. Enzymatical hydrolysis of N-glycans from glycoproteins and fluorescent labeling by 2-aminobenzamide (2-AB). Methods Mol. Biol. 2008, 446, 231–238.

(18) Ahn, J.; Bones, J.; Yu, Y. Q.; Rudd, P. M.; Gilar, M. Separation of 2-aminobenzamide labeled glycans using hydrophilic interaction chromatography columns packed with 1.7 microm sorbent. J. Chromatogr. B 2010, 878, 403−408.

(19) Telford, J. E.; Bones, J.; McManus, C.; Saldova, R.; Manning, G.; Doherty, M.;Leweke, F. M.; Rothermundt, M.; Guest, P. C.; Rahmoune, H.; Bahn, S.; Rudd, P. M. Antipsychotic treatment of acute paranoid schizophrenia patients with olanzapine results in altered glycosylation of serum glycoproteins. J. Proteome Res. 2012, 11, 3743–3752.

(20) Houel S.; Hilliard M.; Yu Y. Q.; Mcloughlin N.; Martin S. M.; Rudd P. M.; Williams J. P.;Chen, W. N- and O-glycosylation analysis of etanercept using liquid chromatography and quadrupole time-of-flight mass spectrometry equipped with electron-transfer dissociation functionality. Anal. Chem. 2014, 86, 576−584.

(21) Gilar, M.; Yu, Y. Q.; Ahn, J.; Xie, H.; Han, H.; Ying, W.; Qian, X. Characterization of glycoprotein digests with hydrophilic interaction chromatography and mass spectrometry.Anal. Biochem. 2011, 417, 80–88.

(22) Wu, S. W.; Pu, T. H.; Viner, R.; Khoo, K. H. Novel LC-MS/MS product dependent parallel data acquisition function and data analysis workflow for sequencing and identification of intact glycopeptides. Anal. Chem. 2014, 86, 5478–5486.

(23) Steelman, S. L.; Pohley, F. M. Assay of the follicle-stimulating hormone based on the augmentation with human chorionic gonadotropin. Endocrinology 1953, 53, 604–616.

(24) Anobile, C. J.; Talbot, J. A.; McCann, S. J.; Padmanabhan, V.; Robertson, W. R.Glycoform composition of serum gonadotrophins through the normal menstrual cycle and in the post-menopausal state. Mol. Hum. Reprod. 1998, 4, 631–639.

(25) Walton, W. J.; Nguyen, V. T.; Butnev, V. Y.; Singh, V.; Moore, W. T.; Bousfield, G. R. Characterization of human FSH isoforms reveals a nonglycosylated beta-subunit in addition to the conventional glycosylated beta-subunit. J. Clin. Endocrinol. Metab. 2001,86, 3675–3685.

(26) Bishop, L. A.; Nguyen, T. V.; Schofield, P. R. Both of the β-subunit carbohydrate residues of follicle-stimulating hormone determine the metabolic clearance rate and in vivo potency. Endocrinology 1995, 136, 2635–2640.

(27) Royle, L.; Radcliffe, C. M.; Dwek, R. A.; Rudd, P. M. Detailed structural analysis of N-glycans released from glycoproteins in SDS-PAGE gel bands using HPLC combined with exoglycosidase array digestions. Methods Mol. Biol. 2006, 347, 125–144.

(28) Noguchi, A.; Mukuria, C. J.; Suzuki, E.; Naiki, M. Immunogenicity of N-glycolylneuraminic acid-containing carbohydrate chains of recombinant human erythropoietin expressed in Chinese hamster ovary cells. J. Biochem. 1995, 117, 59–62.

(29) Baenziger, J. U. Glycoprotein hormone GalNAc-4-sulphotransferase. Biochem. Soc. Trans. 2003, 31, 326–330.

(30) Rose, M. P.; Gaines Das, R. E.; Balen, A. H. Definition and measurement of follicle stimulating hormone. Endocr. Rev. 2000, 21, 5–22.

(31) Andre, S.; Unverzagt, C.; Kojima, S.; Frank, M.; Seifert, J.; Fink, C.; Kayser, K.; von der Lieth, C. W.; Gabius, H. J. Determination of modulation of ligand properties of synthetic complex-type biantennary N-glycans by introduction of bisecting GlcNAc in silico,in vitro and in vivo. Eur. J. Biochem. 2004, 271, 118–134.

(32) Dalpathado, D. S.; Irungu, J.; Go, E. P.; Butnev, V. Y.; Norton, K.; Bousfield G. R.;Desaire, H. Comparative glycomics of the glycoprotein follicle stimulating hormone:glycopeptide analysis of isolates from two mammalian species. biochemistry 2006, 45,8665–8673.

 

 

Figure 1 Glycoproteomic strategy and hFSH glycosylation characterization. (a) Strategy for the comparative assessment of glycosylation in rhFSH and uhFSH. (b) SDS-PAGE and MALDI-TOF mass spectra of both hFSHs. (c) IEF result (pH gradient: 3–6) of both hFSHs. (d) Sialic acid contents (n=2). Error bars represent SD from duplicate determinants. (e) Mass spectrum of non-glycosylation site at FC#ISINTTWC#AGYC#YTR [(M+3H)3+ at m/z 724.98] without the 0.98 Da increase after PNGase F digestion. #

represents alkylation of cysteine.

 

 

Figure 2 Profiling and relative quantification of N-glycans of hFSHs (n=3). (a) HILIC chromatograms of 2-AB-labeled glycans from rhFSH and uhFSH. The structure and relative contents (>2.50%) of glycan are shown and compared. The two or three glycans with highest relative contents are highlighted in red. *The two glycans are present in one peak. (b) Comparison of the N-glycan structures of rhFSH and uhFSH.

 

 

Figure 3 MS2 spectra of 2-AB-labeled N-glycan structures. Diagnostic ions are marked with corresponding fragment structures. (a) NeuGc1NeuAc1HexNAc4Hex5 from rhFSH [(M+2H)2+ at m/z 1180.34]. (b) NeuAc2HexNAc4Hex5SO41 from rhFSH, [(M+Na+H)2+ at m/z 1214.36]. *The NeuAc was dehydrated. (c) and (d) The two isomers of Fuc2NeuAc1HexNAc5Hex5 from uhFSH, [(M+2H)2+ at m/z 1274.48].

 

 

Figure 4 Site-specific characterization and comparison of N-glycosylation of rhFSH and uhFSH. (a) MS2 spectrum of the N-glycopeptide KVEN[+NeuAc2HexNAc4Hex5]HTAC#H,[(M+3H)3+ at m/z 1100.76]. The sequential losses of monosaccharide residues are assigned. (b) Comparative description of the glycans present at αN52 of rhFSH and uhFSH. The N-glycan structures were assigned based on previous reports.1, 32

 

 

Figure 5 Identification of the O-linked glycosylation sites in rhFSH and uhFSH. (a) HCD spectrum of the O-glycopeptide NSC#ELT[+HexNAc2Hex1]NITIAIEK, [(M+4H)4+ at m/z 544.26] of rhFSH. (b) ETD spectrum of the O-glycopeptide NSC#ELT[+HexNAc2Hex1]NITIAIEK, [(M+4H)4+ at m/z 544.26] of uhFSH. The O-glycosylation sites are both in red, boldface and italics. # represents alkylation of cysteine.

 

 

 

 

Figure 1 Glycoproteomic strategy and hFSH glycosylation characterization. (a) Strategy for the comparative assessment of glycosylation in rhFSH and uhFSH. (b) SDS-PAGE and MALDI-TOF mass spectra of both hFSHs. (c) IEF result (pH gradient: 3–6) of both hFSHs. (d) Sialic acid contents (n=2). Error bars represent SD from duplicate determinants. (e) Mass spectrum of non-glycosylation site at FC#ISINTTWC#AGYC#YTR [(M+3H)3+ at m/z 724.98] without the 0.98 Da increase after PNGase F digestion. # represents alkylation of cysteine.266x187mm (150 x 150 DPI)

 

 

 

Figure 5 Identification of the O-linked glycosylation sites in rhFSH and uhFSH. (a) HCD spectrum of the Oglycopeptide NSC#ELT[+HexNAc2Hex1]NITIAIEK, [(M+4H)4+ at m/z 544.26] of rhFSH. (b) ETD spectrum of the O-glycopeptide NSC#ELT[+HexNAc2Hex1]NITIAIEK, [(M+4H)4+ at m/z 544.26] of uhFSH. The Oglycosylation sites are both in red, boldface and italics. # represents alkylation of cysteine. 834x820mm (72 x 72 DPI)