Glycobiology Advance Access originally published online on July 19, 2007
Glycobiology 2007 17(10):1120-1126; doi:10.1093/glycob/cwm076
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The sperm agglutination antigen-1 (SAGA-1) glycoforms of CD52 are O-glycosylated
5 Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, UK
6 M-SCAN Ltd., Wokingham, Berks RG41 2TZ, UK
7 Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA
1 To whom correspondence should be addressed: Tel: +44-207-5945219; Fax: +44-207-2250458; e-mail: a.dell{at}imperial.ac.uk
Received on June 13, 2007; revised on July 9, 2007; accepted on July 10, 2007
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Abstract |
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CD52 is composed of a 12 amino acid peptide with N-linked glycans bound to the single potential glycosylation site at position 3, and a glycosylphosphatidylinositol-anchor attached at the C-terminus. Some glycoforms of this molecule expressed in the male reproductive tract are recognized by complement-dependent sperm-immobilizing antibodies in infertile patients making this antigen an important target for immunocontraception and fertility studies. Although the amount of posttranslational modification is already remarkable for such a small polypeptide, O-glycosylation of CD52 has additionally been implicated by several studies, but never rigorously characterized. In this report, we show clear evidence for the presence of O-glycans in CD52 preparations immunopurified using the murine S19 monoclonal antibody generated against sperm agglutination antigen-1 (SAGA-1), a male reproductive tract specific form of CD52. The O-glycans have been characterized by MALDI-TOF and tandem mass spectrometry after reductive elimination and permethylation. The data indicate that the major SAGA-1 O-glycans are core 1 and 2 mucin-type structures, with and without sialic acid (NeuAc0–2Hex1–3HexNAc1–2HexNAcitol). Minor fucosy- lated O-glycans are also present including some struc- tures with putative Ley epitopes (NeuAc0–1Fuc1–3Hex1–2 HexNAc0–1HexNAcitol). Analysis of O-glycopeptides by tandem mass spectrometry provided an additional level of support for the O-glycosylation of SAGA-1. Elucidation of the O-glycosylation of SAGA-1 adds to the complexity of this molecule and may help to explain its biological activity.
Key words: CD52 / O-glycan / mass spectrometry
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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CD52 is a low molecular weight, GPI-anchored antigen with a single N-linked glycan structure, the expression of which is restricted to the male reproductive tract and leukocytes (Kirchhoff and Schroter 2001
; Norton et al. 2002). Remarkably, the polypeptide backbone of CD52 comprises only 12 amino acids (G-Q-N-D-T-S-Q-T-S-S-P-S). In the mammalian male reproductive tract, CD52 was identified on the surface of mammalian spermatozoa, in seminal plasma, and in the epididymal epithelium (Hale et al. 1993; Rooney et al. 1996; Diekman, Norton, Klotz, Westbrook, and Herr 1999; Norton et al. 2002). CD52 is expressed by the epididymal epithelium and is then secreted with its GPI-anchor intact into the epididymal lumen (Kirchhoff 1996). CD52 is inserted into the sperm plasmalemma during epididymal maturation. CD52 has been termed the major maturation-associated sperm membrane antigen in the rat model because the appearance of sperm-surface CD52 in the epididymis coincides with the appearance of fertilization-related abilities, such as forward motility and zona pellucida binding ability.
Functional studies identified a putative role for lymphocyte CD52 in signal transduction during lymphocyte activation (Valentin et al. 1992; Fabian et al. 1993; Lund-Johansen et al. 1993; Treumann et al. 1995; Kirchhoff 1996; Schröter et al. 1999; Hederer et al. 2000). The function of CD52 in the male reproductive tract and mature spermatozoa remains unknown. However, a significant insight into sperm CD52 function was obtained through the inhibition of sperm–zona pellucida tight binding by Fab fragments of S19 (Mahony et al. 1991), a monoclonal antibody (mAb) specific for a carbohydrate epitope on male reproductive tract CD52 (mrtCD52) that is also referred to as sperm agglutination antigen-1 (SAGA-1). These results suggested that the S19 mAb blocked a portion of a zona-binding protein on the sperm surface and implicated CD52 itself or a CD52-containing complex in sperm–zona interactions.
The structure of CD52 has been extensively characterized. Immunochemical analyses with mAbs specific to carbohydrate epitopes on mrtCD52 indicated structural differences between the N-glycans on mrtCD52 and those on lymphocyte CD52 (Diekman, Norton, Klotz, Westbrook, Shibahara, et al. 1999). Comparison of mass spectrometry analyses confirmed that the lymphocyte CD52 and mrtCD52 N-glycans are structurally distinct. Treumann et al. (1995) identified multiple structures at the single N-glycosylation site in lymphocyte CD52 as tetra- and penta-antennary branched structures with poly-N-acetyllactosamine repeats and variable sialylation of terminal monosaccharide residues. Schröter et al. (1999) demonstrated a more complex array of greater than 50 poten- tial N-glycan structures occurring at the N-glycosylation site in CD52 isolated from seminal plasma. The mrtCD52 N-linked glycans were identified as bi-, tri-, tetra-, penta-, and hexa-antennary structures with poly-N-acetyllactosamine repeats and extensive sialylation of terminal residues. Collectively, these results demonstrate that mrtCD52 and lymphocyte CD52 represent glycoforms, glycoproteins with the same core protein, but with different carbohydrate structures.
Although six serine and threonine residues are present in the mature CD52 peptide, O-linked glycosylation has not received the same attention as the N-linked glycans. To date, lectin blotting has indicated the presence of O-linked glycans on mrtCD52 (Hasegawa et al. 2004; Flori et al. 2005), but no rigorous structural studies have been reported. In this paper, we present clear mass spectrometric evidence for the presence of O-glycans in CD52 preparations immunopurified using the murine S19 mAb generated against SAGA-1, and show the putative structures of these glycans.
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Results |
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MALDI-TOF analysis of released O-glycans from SAGA-1
The SAGA-1 glycoforms of CD52 were obtained by purification from human seminal plasma by a combination of lipid phase partitioning, S19 immunoaffinity, and size exclusion. The sample was treated with PNGase F to release the N-glycans which were subsequently separated from the O-glycopeptides by C18 Sep-Pak chromatography. O-glycans were released from the glycopeptides by reductive elimination and their permethyl derivatives were analyzed by MALDI-TOF mass spectrometry (Figure 1). Pseudomolecular ions corresponding to the molecular weight of known O-glycan structures were observed. Two of the most intense ions are at m/z 983 and 1344 which correspond to glycans with the compositions Hex2HexNAc1HexNAcitol and NeuAc1Hex2HexNAc1HexNAcitol, respectively. LacNAc extentions of these two glycans are also present at m/z 1432 (Hex3HexNAc2HexNAcitol) and 1793 (NeuAc1Hex3HexNAc2 HexNAcitol). A major signal at m/z 534 has a composition of Hex1HexNAcitol which is consistent with a short core 1 type structure. Mono- and di-sialylated versions of this glycan are also indicated by ions at m/z 895 and 1256, respectively. Fucosylated versions of the major ions at m/z 534, 983, and 1344 were observed at m/z 708, 1157, and 1518, respectively. Ions consistent with glycans carrying multiple fucose residues were detected in very low amounts at m/z 1505 and 1692.
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) Galactose; (
) GlcNAc; (
) Fucose; (
) NeuAc; (
R) GalNAcitol.Tandem mass spectrometry analysis of SAGA-1 O-glycans defines sequences
Each of the components observed in the MALDI-TOF experiment was subjected to tandem mass spectrometry in order to assign sequences. To avoid co-selection of matrix ions which are abundant in the m/z range below 900 in the MALDI-TOF/TOF, CAD-ESI-MS/MS was used to analyze the low molecular weight components at m/z 534 and 895 (NeuAc0–1Hex1HexNAcitol). These are core type 1 O-glycans as indicated by the presence of a signal at m/z 298 (data not shown) corresponding to the elimination of NeuAcHex from the 3-position of HexNAcitol (Kui Wong et al. 2003
). The remaining larger O-glycans were analyzed by MALDI-TOF/TOF and gave diagnostic fragment ions that supported their assignments in Figure 1. Representative data from the component at m/z 1344 (NeuAc1Hex2HexNAc1HexNAcitol) in the MALDI-TOF spectrum (Figure 1) are shown in Figure 2A. The fragment ion at m/z 284 indicates that the glycan is predominantly core 2 type (see inset to Figure 2A). The glycan at m/z 1344 is predicted to contain a single NeuAc based on its composition. The spectrum in Figure 2A suggests that the NeuAc residue can be added to either antenna, as fragment ions at m/z 520, 847, and 1108 support one isomer and the ion at m/z 747 supports another. The MALDI-TOF/TOF spectrum of the minor component at m/z 1505 is also shown (Figure 2B). Although the signals are weak, many diagnostic ions were observed which are consistent with a tri-fucosylated O-glycan containing a Ley epitope (see inset to Figure 2B).
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ESI-MS and CAD-ESI-MS/MS of HF products show that SAGA-1 is O-glycosylated
Although the data above demonstrate the presence of O-glycans in the SAGA-1 preparation, glycoproteomics data, where O-glycans are still attached to the polypeptide provide an additional level of confidence for O-glycosylation. To achieve this, PNGase F-treated material was purified by Sep-Pak chromatography and subjected to mild hydrofluoric acid (HF) hydrolysis to release the peptide from the GPI anchor. The HF-treated peptide was analyzed by ESI-MS and CAD-ESI-MS/MS. ESI-MS gave doubly charged ions at m/z 8092+ and 9922+ consistent with the presence of the peptide GQDDTSQTSSPS (Asn at position 3 is converted to Asp when the N-glycans were released by PNGase F) substituted with ethanolamine at the C-terminus and carrying glycans of compositions Hex1HexNAc1 and Hex2HexNAc2, respectively. CAD-ESI-MS/MS of m/z 8092+ and 9922+ (Figure 3A and B, respectively) gave diagnostic signals for sugar fragments at m/z 204 and 366 which correspond to HexNAc and HexHexNAc, respectively. Peptide and glycopeptide signals for each parent ion are also present (see annotations in Figure 3). Notably, m/z 1252 corresponds to loss of the intact glycan chain from each molecular ion, while the signals at m/z 7282+ (Figure 3A), 1455, and 1617 (Figure 3B) correspond to partial loss of the glycan.
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O-Glycan structural conclusions
Taking into consideration the MALDI-TOF and the MS/MS data, we conclude that (i) SAGA-1 glycoforms of CD52 are O-glycosylated with a heterogeneous mixture of glycans ranging from two to seven monosaccharide residues (see Figure 4); (ii) both core type 1 and core type 2 O-glycans are present; (iii) many of the O-glycans are sialylated, carrying a maximum of two sialic acids, with the remaining O-glycans being noncapped; (iv) fucosylation is present at relatively low levels and putative Ley epitopes are present as well as mono-fucosylated glycans; (v) some glycans carry a lacNAc extension on the 6-arm of the core 2 structure.
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N-Glycan structural conclusions
To ensure that the SAGA-1 glycoforms of CD52 are representative of this class of molecule and not the result of aberrant processing, the N-glycans of SAGA-1 were released, permethylated and analyzed by MALDI-TOF (supplementary Figure 1). The results show a series of ions that are consistent with highly sialylated multi-antennary glycans with varying numbers of polylactosamine repeats. This agrees with the structures reported previously (Schröter et al. 1999
). To confirm the presence of polylactosamine repeats, released N-glycans were digested with endo-ß-galactosidase, an enzyme that cleaves on the reducing end side of internal galactose sugars (Fukuda and Matsumura 1976). The digestion products were permethylated, purified by Sep-Pak chromatography and analyzed by MALDI-TOF (supplementary Figure 2). Truncated complex N-glycans were observed at m/z 1836, 2081, and 2326 consistent with the loss of two, three, and four antennae, respectively. Other complex N-glycans were also truncated, but carried one or more sialylated antenna(e) that was resistant to digestion (e.g., m/z 2891 and 3456). The ions at m/z 1084 and 1445 correspond to digestion products from antennae and indicate that antennae with poly-N-acetyllactosamine repeats are often sialylated.
In summary, the data from the MALDI profiling complemented by the endo-ß-galactosidase digestion indicated that the N-glycans of the SAGA-1 preparation are not observably different from those characterized by Schröter and colleagues (1999) who examined total CD52 preparations from seminal plasma.
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Discussion |
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The lymphocyte and male reproductive tract versions of CD52 share the same 12 amino acid peptide, yet the glycosylation of the two molecules is quite distinct (Diekman et al. 1999
; Schröter et al. 1999). Indeed, monoclonal antibodies raised against spermatozoa, that recognize glycan epitopes on CD52, do not cross-react with the lymphocyte form of CD52 (Kameda et al. 1992; Diekman, Norton, Klotz, Westbrook, Shibahara, et al. 1999; Norton et al. 2002) and have sperm-agglutinating and complement-dependent sperm-immobilization activities in vitro (Isojima et al. 1987; Diekman, Norton, Klotz, Westbrook, Shibahara, et al. 1999). Expression of such antibodies in female sera or reproductive tracts is associated with reduced fertility (Ohl and Naz 1995; Kutteh et al. 1996), so understanding of the structure of CD52 has implications for both fertility treatments as well as contraception.
Although the N-glycans of CD52 have been characterized in detail, the O-glycans have proven more elusive. To date, lectin studies and the shift in mass of CD52 following reductive elimination have been used to indicate that O-glycans might be present (Hasegawa et al. 2004; Flori et al. 2005), but no structural analysis has been performed. In this study, we have used mass spectrometry to conclusively demonstrate O-glycosylation of CD52 and have provided a profile of putative structures. Figure 5 shows a schematic of SAGA-1 and demonstrates the remarkable complexity of the molecule derived from its posttranslational modifications.
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MALDI-TOF profiling of the permethylated SAGA-1 O-glycans revealed a number of ions corresponding to core 1 and core 2 structures (Figure 1). Tandem mass spectrometry analysis of the ions confirmed the presence of these glycans and enabled structural predictions to be made (Figure 2). Consistent with the high levels of sialylation observed in the male-specific CD52 N-glycans (Schröter et al. 1999
), sialic acid was a significant terminal feature of the O-glycans. These charged moieties may contribute to the formation of a glycocalyx adjacent to the sperm membrane, and may prevent nonspecific binding of the spermatozoon to other spermatozoa and/or the reproductive tract epithelium (Schröter et al. 1999).
Amongst the pseudomolecular ions observed in the MALDI-TOF profile, several minor signals were observed that were consistent with fucosylated glycans (m/z 708, 1157, 1505, 1518, and 1692 in Figure 1). MALDI-TOF/TOF analysis provided support for the fucosylation of these glycans. The presence of fucosylated glycans is not surprising. Core fucosylation and some peripheral 
1,3-linked fucose residues were reported in the N-glycans of male-specific CD52 (Schröter et al. 1999). Interestingly, the peripheral fucose in the N-glycans formed a VIM2 epitope (NeuAc2-3Galß1-4GlcNAcß1-3Galß1-4(Fuc1-3)GlcNAc), whereas, the putative O-glycan at m/z 1157 would form a Lea (Galß1-3(Fuc1-4)GlcNAc) or Lex (Galß1-4(Fuc1-3)GlcNAc) epitope. As with the N-glycans, no evidence for sialyl Lea/x was observed. Lex and Ley (Fuc1-2Galß1-4(Fuc1-3)GlcNAc) sequences are known to be abundant in seminal plasma and have been linked with a role in sperm capacitation/decapacitation (Morris et al. 1996; Chalabi et al. 2002; Piludu et al. 2007). Glycodelin-S, a human seminal glycoprotein that carries Lex and Ley epitopes, significantly increases sperm binding to the hemizona assay at physiological conditions (Morris et al. 1996). The putative immunosuppressive properties of the Lex/y epitopes of Glycodelin-S may also mediate the low immunogenicity of spermatozoa in women (Morris et al. 1996).
Additional evidence for the O-glycosylation of CD52 from human seminal plasma was obtained by tandem mass spectrometry of the glycoconjugate following HF and PNGase F treatment which removes the GPI anchor and the N-glycans, respectively. The spectra contained strong ions at m/z 204 and 366 (Figure 3) which are diagnostic for glycans, since glycosidic linkages are more susceptible to fragmentation than peptide bonds. The completely deglycosylated form of the peptide-ethanolamine molecule (m/z 1252) was also observed in both spectra, further supporting the structural assignments. Together with ions corresponding to fragmentation of the glycan component of the glycopeptide (m/z 7282+ in Figure 3A and m/z 1455 and 1617 in Figure 3B), these data provide firm evidence for O-linked glycosylation of the SAGA-1 polypeptide.
Although several ions derived from peptide bond breakage were observed in the tandem mass spectrometry spectra of the glycopeptides (Figure 3), the amount of material was insufficient to sequence through the site(s) of O-glycosylation. Previously, Diekman et al. (Diekman, Norton, Klotz, Westbrook, Shibahara, et al. 1999) used Edman degradation to sequence SAGA-1, an immunopurified version of CD52, and reported a blank cycle at the threonine at position 8, leading to the conclusion that this residue was one of the sites of O-glycosylation. Similar studies were undertaken by two other groups and additional sites of O-glycosylation have been reported at positions Thr-5 (Flori et al. 2005) and Thr-5, Ser-6, Ser-9 and Ser-10 (Hasegawa et al. 2003), indicating there may be more than one site of attachment.
In conclusion, this report represents another important milestone in the elucidation of the molecular structure of the biologically important antigen CD52. Examining an immunopurified form of CD52 from human seminal plasma, we used high sensitivity mass spectrometry to characterize for the first time the O-glycans of this molecule.
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Materials and methods |
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Purification of SAGA-1 from human seminal plasma
Studies involving human semen donors were approved by the University of Virginia's Human Research Committee and informed consent was obtained from each participant after explanation of the nature and possible consequences of the studies. Human seminal plasma was prepared by clarifying pooled human semen samples by centrifugation to remove cellular components. Lipid phase partitioning, as described by Svennerholm and Fredman (1980
) was used as the first step in the purification of SAGA-1 from seminal plasma. To extract lipid-bound material, seminal plasma was adjusted to 4:8:3 of chloroform/methanol/aqueous sample and centrifuged at 12000 x g to remove insoluble components. The chloroform/methanol/water ratio of the recovered supernatant was adjusted to 4:8:5.6, and the mixture was allowed to separate into a lower chloroform phase and an upper aqueous phase. The aqueous phase containing SAGA-1 was concentrated by evaporation under vacuum to remove organic solvent and resuspended in column binding buffer (0.05% deoxycholate, 20 mM sodium phosphate, pH 7.4). SAGA-1 was immunoaffinity purified on immobilized S19 mAb (Diekman et al. 1997) that had been prepared by coupling the S19 mAb to Sepharose 4B (Amersham Biosciences, Piscataway, NJ) according the manufacturer's instructions. Bound material was eluted with 0.5% deoxycholate, 50 mM triethylamine, pH 11.5, and neutralized with 500 mM sodium phosphate, pH 7.0. Eluted fractions containing SAGA-1 were pooled and subjected to size exclusion on a Bio-Rad Prep Cell Model 491 (Bio-Rad, Hercules, CA). Purity was assessed by SYPRO Ruby protein stain (Bio-Rad) and S19 immunoblot analysis, and purified SAGA-1 was utilized for carbohydrate structural characterization.
PNGase F digestion of glycopeptides
PNGase F (EC 3.5.1.5
[EC]
2, Roche Molecular Biochemicals, Lewes, UK) digestion was carried out in ammonium bicarbonate (50 mM, pH 8.5) for 16 h at 37°C using 3 U of enzyme. The reaction was terminated by lyophilization and the released N-glycans were separated from peptides and O-glycopeptides by Sep-Pak C18 (Waters, Elstree, UK) as described (Dell et al. 1993).
endo-ß-Galactosidase digestion of N-glycans
endo-ß-Galactosidase (Seikagaku, Tokyo, Japan) digestion was carried out in ammonium acetate (50 mM, pH 5.5) for 18 h at 37°C using 10 mU of enzyme. The reaction was terminated by lyophilization.
Reductive elimination
O-Glycans were released by reductive elimination which was performed in 400 µL of potassium borohydride (54 mg/mL in 0.1 M potassium hydroxide) at 45°C. The reaction was carried out for 16 hours and terminated by dropwise addition of glacial acetic acid, followed by Dowex chromatography and borate removal (Dell et al. 1993, 1994).
HF hydrolysis
Samples were incubated with 50 µL of 48% HF (Sigma, Poole, UK) on ice for 20 h and the reagent removed by drying down under a gentle stream of nitrogen.
Derivatization for MALDI-TOF and tandem mass spectrometry analysis
Permethylation was performed using the sodium hydroxide procedure, as described previously (Dell et al. 1993).
Mass spectrometric analysis
MALDI-TOF data were acquired on a Voyager-DE sSTR mass spectrometer (PerSeptive Biosystems, Framingham, MA) in the reflectron mode with delayed extraction. Permethylated samples were dissolved in 10 µL of 80% (v/v) methanol in water, and 1 µL of dissolved sample was premixed with 1 µL of matrix (10 mg/mL 2,5-dihydroxybenzoic acid (DHB) in 80% (v/v) aqueous methanol) before loading onto a metal plate.
CAD ESI-MS/MS spectra were acquired using a Q-STAR (Applied Biosystems, Foster City, CA) mass spectrometer. The permethylated glycans were dissolved in methanol before loading into a spray capillary (Proxeon Biosystems, Odense, Denmark), coated with a thin layer of gold/palladium, in a final volume of 2 µL. A potential of 1.5 kV was applied to a nanoflow tip to produce a flow rate of 10–30 nL/min. The drying gas used for the instrument was N2 and the collision gas was argon, with the collision gas pressure maintained at 10–4 millibar. Collision energies varied depending on the size of the carbohydrate, typically between 30 and 90 eV. The MALDI-TOF/TOF experiments were performed on a 4800 Proteomics Analyzer (Applied Biosystems, Foster City, CA) operated in reflectron positive ion mode. Both DHB (for glycans) and -cyano-4-hydroxycinnamic acid (for peptide standards) matrices (10 mg/mL in 50% (v/v) acetonitrile in 0.1% (v/v) aqueous trifluoroacetic acid) were used in conjunction with setting the potential difference between the source acceleration voltage and the collision cell at 1 kV to obtain different degrees and patterns of fragmentation.
Supplementary data for this article is available online at www.glycob.oxfordjournals.org.
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Funding |
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Biotechnology and Biological Sciences Research Council (BBSRC) to A.D., H.R.M, and S.M.H; National Institutes of Health (U54 HD 29099, R01 HD 35523 to J.C.H). AD is a BBSRC Professorial Research Fellow.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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None declared.
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Footnotes |
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2 Present address: School of Science and Technology, University Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia.
3 Present address: M-SCAN Ltd., Wokingham, Berks RG41 2TZ, UK.
4 Present address: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
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Abbreviations |
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CAD, collisionally-activated dissociation; ESI, electrospray ionization; GPI, glycosylphosphatidylinositol; HF, hydrofluoric acid; mAb, monoclonal antibody; MALDI, matrix-assisted laser desorption/ionization; mrt, male reproductive tract; MS, mass spectrometry; MS/MS, tandem mass spectrometry; PNGase F, peptide N-glycosidase F; SAGA-1, sperm agglutination antigen-1; TOF, time-of-flight.
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