Identification of Bovine Sperm Surface Proteins Involved in Carbohydrate-mediated Fertilization Interactions
2016; Elsevier BV; Volume: 15; Issue: 7 Linguagem: Inglês
10.1074/mcp.m115.057703
ISSN1535-9484
AutoresSira Defaus, Manuel Avilés, David Andreu, Ricardo Gutiérrez‐Gallego,
Tópico(s)Animal Genetics and Reproduction
ResumoGlycan-protein interactions play a key role in mammalian fertilization, but data on the composition and identities of protein complexes involved in fertilization events are scarce, with the added complication that the glycans in such interactions tend to differ among species. In this study we have used a bovine model to detect, characterize and identify sperm lectins relevant in fertilization. Given the complexity of the sperm-toward-egg journey, two important aspects of the process, both primarily mediated by protein-sugar interactions, have been addressed: (1) formation of the sperm reservoir in the oviductal epithelium, and (2) gamete recognition (oocyte-sperm interaction). Using whole sperm cells and a novel affinity capture method, several groups of proteins with different glycan specificities, including 58 hitherto unreported as lectins, have been identified in sperm surface, underscoring both the efficacy of our selective approach and the complex composition and function of sperm. Based on these results and previous data, we suggest that sperm surface proteins play significant roles in fertilization events such as membrane remodeling, transport, protection and function, thus supporting the hypothesis that rather than a simple lock-and-key model, mammalian fertilization relies on a complex interactome involving multiple ligands/receptors and recognition/binding events. Glycan-protein interactions play a key role in mammalian fertilization, but data on the composition and identities of protein complexes involved in fertilization events are scarce, with the added complication that the glycans in such interactions tend to differ among species. In this study we have used a bovine model to detect, characterize and identify sperm lectins relevant in fertilization. Given the complexity of the sperm-toward-egg journey, two important aspects of the process, both primarily mediated by protein-sugar interactions, have been addressed: (1) formation of the sperm reservoir in the oviductal epithelium, and (2) gamete recognition (oocyte-sperm interaction). Using whole sperm cells and a novel affinity capture method, several groups of proteins with different glycan specificities, including 58 hitherto unreported as lectins, have been identified in sperm surface, underscoring both the efficacy of our selective approach and the complex composition and function of sperm. Based on these results and previous data, we suggest that sperm surface proteins play significant roles in fertilization events such as membrane remodeling, transport, protection and function, thus supporting the hypothesis that rather than a simple lock-and-key model, mammalian fertilization relies on a complex interactome involving multiple ligands/receptors and recognition/binding events. Fertilization is a fundamental event that follows a highly coordinated sequence of cellular interactions between gametes in order to form a diploid zygote and, ultimately, a new individual. There is now considerable evidence that carbohydrate recognition plays a major role in fertilization, from lower species to man (1Dell A. Morris H.R. Easton R.L. Patankar M. Clark G.F. The glycobiology of gametes and fertilization.Biochim. Biophys. Acta. 1999; 1473: 196-205Crossref PubMed Scopus (82) Google Scholar, 2Pang P.C. Chiu P.C. Lee C.L. Chang L.Y. Panico M. Morris H.R. Haslam S.M. Khoo K.H. Clark G.F. Yeung W.S. Dell A. Human sperm binding is mediated by the sialyl-Lewis(x) oligosaccharide on the zona pellucida.Science. 2011; 333: 1761-1764Crossref PubMed Scopus (250) Google Scholar), and also that some sperm-surface carbohydrates are implicated in immune-mediated infertility (3Diekman A.B. Glycoconjugates in sperm function and gamete interactions: how much sugar does it take to sweet-talk the egg?.Cell Mol. Life Sci. 2003; 60: 298-308Crossref PubMed Scopus (73) Google Scholar). In this context, it is well established that oviductal sperm reservoir formation (4Suarez S.S. Carbohydrate-mediated formation of the oviductal sperm reservoir in mammals.Cells Tissues. Organs. 2001; 168: 105-112Crossref PubMed Scopus (84) Google Scholar) and gamete recognition (5Clark G.F. The role of carbohydrate recognition during human sperm-egg binding.Hum. Reprod. 2013; 28: 566-577Crossref PubMed Scopus (56) Google Scholar) in mammals are predominantly mediated by protein-carbohydrate interactions involving, on the one hand, sugar moieties on both the oviductal epithelium and the oocyte zona pellucida (ZP) 1The abbreviations used are:ZPzona pellucidaAALAleuria aurantia lectinAGCauto gain controlCMcapacitating mediumCRDcarbohydrate recognition domainCREDEXCarbohydrate REcognition Domain EXcisionDDAdata dependent acquisitionDVSdivinylsulfoneFucfucoseFuc4NFucα1–4GlcNAcGalgalactoseGlcglucoseGlcNAcN-acetylglucosamineGOgene ontologyLewis A(Lea) Galβ1–3(Fucα1–4)GlcNAcLTALotus tetragonolobus agglutininLTQlinear trap quadrupoleMmolar - mol per literMAAMaackia amurensis agglutininmMmillimolarMVSmethyl vinylsulfoneNCMnoncapacitating mediumNeu5AcN-acetylneuraminic acidppmparts-per-millionrpmrevolutions per minuteRTroom temperatureSiasialic acidSialyl-Lewis X (SLex)Neu5Acα2–3Galβ1–4(Fucα1–3)GlcNAcSNsupernatantSNASambucus nigra agglutininTristris(hydroxymethyl)aminomethaneUEA-IUlex europaeus agglutinin I3′-SLNNeu5Acα2–3Galβ1–4GlcNAc. and, on the other hand, carbohydrate-binding proteins (i.e. lectins) on the sperm cell surface. Different candidate molecules potentially involved in sperm-oviduct and/or sperm-egg interactions have been postulated for various mammalian species (6Suarez S.S. Mammalian sperm interactions with the female reproductive tract.Cell Tissue Res. 2016; 363: 185-194Crossref PubMed Scopus (172) Google Scholar, 7Töpfer-Petersen E. Molecules on the sperm's route to fertilization.J. Exp. Zool. 1999; 285: 259-266Crossref PubMed Scopus (40) Google Scholar), suggesting that different carbohydrate ligands and lectins are involved in each case. However, no consensus still exists on the binding mechanisms and the molecules therein involved, mainly due to the incomplete identification of all players. Specifically, in the bovine species, it has been demonstrated that sperm binding to the oviductal epithelium involves fucose (Fuc) recognition (8Lefebvre R. Lo M.C. Suarez S.S. Bovine sperm binding to oviductal epithelium involves fucose recognition.Biol. Reprod. 1997; 56: 1198-1204Crossref PubMed Scopus (157) Google Scholar, 9Suarez S.S. Revah I. Lo M. Kolle S. Bull sperm binding to oviductal epithelium is mediated by a Ca2+-dependent lectin on sperm that recognizes Lewis-a trisaccharide.Biol. Reprod. 1998; 59: 39-44Crossref PubMed Scopus (79) Google Scholar) and that, following capacitation, spermatozoa are released from the reservoir and proceed further into the oviduct to meet the oocyte, the gamete interaction being predominantly mediated by sialic acid (Sia) residues on the oocyte (10Velásquez J.G. Canovas S. Barajas P. Marcos J. Jiménez-Movilla M. Gallego R.G. Ballesta J. Avilés M. Coy P. Role of sialic acid in bovine sperm-zona pellucida binding.Mol. Reprod Dev. 2007; 74: 617-628Crossref PubMed Scopus (54) Google Scholar). zona pellucida Aleuria aurantia lectin auto gain control capacitating medium carbohydrate recognition domain Carbohydrate REcognition Domain EXcision data dependent acquisition divinylsulfone fucose Fucα1–4GlcNAc galactose glucose N-acetylglucosamine gene ontology (Lea) Galβ1–3(Fucα1–4)GlcNAc Lotus tetragonolobus agglutinin linear trap quadrupole molar - mol per liter Maackia amurensis agglutinin millimolar methyl vinylsulfone noncapacitating medium N-acetylneuraminic acid parts-per-million revolutions per minute room temperature sialic acid Neu5Acα2–3Galβ1–4(Fucα1–3)GlcNAc supernatant Sambucus nigra agglutinin tris(hydroxymethyl)aminomethane Ulex europaeus agglutinin I Neu5Acα2–3Galβ1–4GlcNAc. To gain insights into gamete events associated with fertilization (oviduct adhesion, capacitation, ZP binding and acrosomal exocytosis), analyzing the sperm surface proteome is a must (11Brewis I.A. Gadella B.M. Sperm surface proteomics: from protein lists to biological function.Mol. Hum. Reprod. 2010; 16: 68-79Crossref PubMed Scopus (83) Google Scholar). Traditionally, sperm surface proteins have been studied using labeling strategies with membrane-impermeable tags to facilitate enrichment and identification (12Shetty J. Naaby-Hansen S. Shibahara H. Bronson R. Flickinger C.J. Herr J.C. Human sperm proteome: immunodominant sperm surface antigens identified with sera from infertile men and women.Biol. Reprod. 1999; 61: 61-69Crossref PubMed Scopus (109) Google Scholar, 13Holt W.V. Elliott R.M. Fazeli A. Satake N. Watson P.F. Validation of an experimental strategy for studying surface-exposed proteins involved in porcine sperm-oviduct contact interactions.Reprod. Fertil. Dev. 2005; 17: 683-692Crossref PubMed Scopus (17) Google Scholar). These approaches are not entirely plasma membrane-proof, as some intracellular proteins may also be accidentally labeled during preparation and experimentation, or endogenous sperm proteins may co-purify with labeled ones. As an alternative to surface labeling strategies, sperm cell plasma membrane fractions can be purified. Some of these techniques, however, tend to give low purifications and poorly defined fractions, or involve treatments that denature proteins, inhibit enzyme activity, or affect the functional integrity of the membranes. Careful evaluation of the strategy is especially relevant for proteins involved in ZP recognition; if the preparation contains acrosomal contamination, intra-acrosomal ZP-binding proteins will be identified that may mask primary (plasma membrane) ZP-binding proteins (14Flesch F.M. Wijnand E. van de Lest C.H. Colenbrander B. van Golde L.M. Gadella B.M. Capacitation dependent activation of tyrosine phosphorylation generates two sperm head plasma membrane proteins with high primary binding affinity for the zona pellucida.Mol. Reprod. Dev. 2001; 60: 107-115Crossref PubMed Scopus (50) Google Scholar). An alternative to subcellular fractionation is enrichment in protein types from a whole cell lysate. For instance, sperm phosphoproteomics studies often resort to affinity-based approaches where enrichment in phosphorylated peptides is achieved on immobilized metal ion (or TiO2) columns (15Platt M.D. Salicioni A.M. Hunt D.F. Visconti P.E. Use of differential isotopic labeling and mass spectrometry to analyze capacitation-associated changes in the phosphorylation status of mouse sperm proteins.J. Proteome Res. 2009; 8: 1431-1440Crossref PubMed Scopus (58) Google Scholar). Another relevant PTM, S-nitrosylation, has been characterized in humans using a biotin switch assay for protein enrichment that provided novel insights on the role of nitric oxide in capacitation (16Lefièvre L. Chen Y. Conner S.J. Scott J.L. Publicover S.J. Ford W.C. Barratt C.L. Human spermatozoa contain multiple targets for protein S-nitrosylation: an alternative mechanism of the modulation of sperm function by nitric oxide?.Proteomics. 2007; 7: 3066-3084Crossref PubMed Scopus (134) Google Scholar). It is also possible to combine subcellular fractionation and protein enrichment; in sperm, the best example is the use of nitrogen cavitation to produce a cytosolic fraction that, after enrichment by poly-Glu:Tyr affinity chromatography, enabled the isolation and identification of four tyrosine kinases specifically localized to the cell cytosol (17Lalancette C. Faure R.L. Leclerc P. Identification of the proteins present in the bull sperm cytosolic fraction enriched in tyrosine kinase activity: a proteomic approach.Proteomics. 2006; 6: 4523-4540Crossref PubMed Scopus (45) Google Scholar). Alternatively, immobilized lectins are used in affinity chromatography to extract surface glycoproteins (18Runnebaum I.B. Schill W.B. Töpfer-Petersen E. ConA-binding proteins of the sperm surface are conserved through evolution and in sperm maturation.Andrologia. 1995; 27: 81-90Crossref PubMed Scopus (27) Google Scholar), a method that can also be employed on nitrogen-cavitated and solubilized sperm plasma membrane material. Finally, sperm head plasma membrane proteins with high primary ZP binding affinity have been specifically isolated using ZP fragment columns (14Flesch F.M. Wijnand E. van de Lest C.H. Colenbrander B. van Golde L.M. Gadella B.M. Capacitation dependent activation of tyrosine phosphorylation generates two sperm head plasma membrane proteins with high primary binding affinity for the zona pellucida.Mol. Reprod. Dev. 2001; 60: 107-115Crossref PubMed Scopus (50) Google Scholar, 19Ensslin M. Calvete J.J. Thole H.H. Sierralta W.D. Adermann K. Sanz L. Töpfer-Petersen E. Identification by affinity chromatography of boar sperm membrane-associated proteins bound to immobilized porcine zona pellucida. Mapping of the phosphorylethanolamine-binding region of spermadhesin AWN.Biol. Chem. Hoppe Seyler. 1995; 376: 733-738Crossref PubMed Scopus (53) Google Scholar). Herein, we apply a novel affinity capture method using immobilized carbohydrates and combining proteolysis of protein-glycan complexes and mass spectrometry (CREDEX-MS, "Carbohydrate REcognition Domain EXcision Mass Spectrometry") (20Przybylski M. Moise A. Siebert H. Gabius H. CREDEX-MS: Molecular elucidation of carbohydrate recognition peptides in lectins and related proteins by proteolytic excision-mass spectrometry.J. Pept. Sci. 2008; 14: 40Google Scholar, 21Przybylski, M., Moise, A., and Gabius, H., (2009) Identification of ligand recognition domains. EP2009/003495. WO/2009/138250,Google Scholar, 22Moise A. Andre S. Eggers F. Krzeminski M. Przybylski M. Gabius H.J. Toward bioinspired galectin mimetics: identification of ligand-contacting peptides by proteolytic-excision mass spectrometry.J. Am. Chem. Soc. 2011; 133: 14844-14847Crossref PubMed Scopus (33) Google Scholar, 23Jiménez-Castells C. Defaus S. Moise A. Przbylski M. Andreu D. Gutiérrez-Gallego R. Surface-based and mass spectrometric approaches to deciphering sugar-protein interactions in a galactose-specific agglutinin.Anal. Chem. 2012; 84: 6515-6520Crossref PubMed Scopus (21) Google Scholar) to examine complex samples of bovine sperm under single sided physiological conditions. In our approach, entire sperm cells rather than solubilized sperm proteins, whole cell lysate, or subcellular fractions were used to eliminate treatment-related uncertainties and to preserve as much as possible the native 3D architecture of sperm surface proteins, essential for ZP binding (24Redgrove K.A. Anderson A.L. Dun M.D. McLaughlin E.A. O'Bryan M.K. Aitken R.J. Nixon B. Involvement of multimeric protein complexes in mediating the capacitation-dependent binding of human spermatozoa to homologous zonae pellucidae.Dev. Biol. 2011; 356: 460-474Crossref PubMed Scopus (75) Google Scholar). In this way, 94 carbohydrate-binding sperm surface proteins addressing four different glycotopes were enriched, identified by state-of-the-art proteomics, and mapped with the fertilization events in bovine species. Of these, 58 proteins had not been previously found in bovine sperm by proteomics approaches, which suggests they represent low-abundance lectins that could only be identified by our selective methodology. Furthermore, a comparison of the proteins identified under capacitating and noncapacitating media (CM and NCM) conditions showed differences in number and composition, demonstrating that the sperm membrane undergoes changes during capacitation, in preparation for downstream functions during fertilization. In addition, correlation of the present bull sperm results with recently published identifications in human sperm proteomes 2The term "sperm proteome," although broadly used, tends to overlook the fact that sperm, depending on its various stages of maturation in both male and female genital tract exhibits quite different proteomic signatures (e.g., [25Skerget S. Rosenow M.A. Petritis K. Karr T.L. Sperm proteome maturation in the mouse epididymis.PLoS ONE. 2015; 10: e0140650Crossref PubMed Scopus (74) Google Scholar]). It has been suggested that a more accurate description of the material here used would be "sperm component of the male ejaculatome." revealed some potentially novel bovine sperm proteins, corroborating the importance of species-specific reproductive biology characterization. Fresh semen was obtained after electroejaculation of bulls (Asturiana de los Valles breed, Bos taurus) at the Cenero (Asturias, Spain) artificial insemination facility. Semen aliquots were stored in liquid nitrogen immediately after collection and kept at −196 °C during transportation and storage. Glycotopes Lea, 3′-SLN and SLex were obtained from Dextra (Reading, UK); Fuc4N was from Toronto Research Chemicals (Toronto, Canada). Lectins LTA, MAA, SNA and UEA-I were from Sigma-Aldrich (Madrid, Spain); AAL was from Vector Labs (Burlingame, CA). Sequencing-grade trypsin was from Promega (Madison, WI, USA). 10 and 30 kDa Amicon centrifugal filters were from Merck Millipore (Madrid, Spain); Mobicol F microcolumns fitted with 35 μm filters were from MoBiTec (Göttingen, Germany). Sepharose, DVS, MVS and other chemicals were from Sigma-Aldrich. Prior to use, semen aliquots were thawed for 10 s at room temperature and immediately afterward placed in a water bath at 37 °C for 40 s. Liquefied semen was next subjected to the washing swim-up technique. Briefly, samples (250 μl aliquots) were layered in cryotube vials under 1 ml of either capacitating (26Parrish J.J. Susko-Parrish J. Winer M.A. First N.L. Capacitation of bovine sperm by heparin.Biol. Reprod. 1988; 38: 1171-1180Crossref PubMed Scopus (1293) Google Scholar) (CM: 114 mm NaCl, 3.2 mm KCl, 0.3 mm NaH2PO4·H2O, 10 mm sodium lactate, 2 mm CaCl2·2H2O, 0.5 mm MgC12·6H2O, 10 mm HEPES, 25 mm NaHCO3, adjusted 0.06% BSA, 1 mm sodium pyruvate, 50 μg/ml gentamicin, pH 7.3) or noncapacitating medium (27Kasekarn W. Kanazawa T. Hori K. Tsuchiyama T. Lian X. Garenaux E. Kongmanas K. Tanphaichitr N. Yasue H. Sato C. Kitajima K. Pig sperm membrane microdomains contain a highly glycosylated 15–25-kDa wheat germ agglutinin-binding protein.Biochem. Biophys. Res. Commun. 2012; 426: 356-362Crossref PubMed Scopus (7) Google Scholar) (NCM: 100 mm NaCl, 0.36 mm NaH2PO4·2H2O, 8.6 mm KCl, 23 mm HEPES, 0.5 mm MgCl2·6H2O, 11 mm glucose, pH 7.6) (both media previously conditioned at 37 °C), and incubated for 1 h at 37 °C. During this time sperm are allowed to swim up in the medium, with the purpose of collecting the most motile, active and normal ones, free of debris and seminal plasma. The supernatant (∼700 μl) was collected and centrifuged at 200 × g for 10 min. The top layer was discarded and the final pellet (∼500 μl) kept at 37 °C for subsequent affinity chromatography experiments. Samples prepared in this way were examined under light microscope before use, and sperm motility and morphology were evaluated. Total sperm count was assessed using an improved Neubauer hemacytometer. A semen aliquot was thawed, divided into two fractions (CM or NCM, respectively; three replicates per fraction), and each sample submitted to the above swim-up procedure. Final pellets obtained under either condition were resuspended in 85 μl of 25 mm NH4HCO3 (pH 8.5), treated with 15 μl of 1 g/L trypsin (Promega, Madison, WI) in NH4HCO3 and incubated overnight at 37 °C; then filtered (Amicon 10 kDa) to remove remaining trypsin and sperm heads and/or tails. The filtrates were lyophilized before LC-MS/MS analysis. For carbohydrate immobilization, 5 mg of each glycotope (Fuc4N, Lea, 3′-SLN and SLex) dissolved in 50 μl of 0.5 m K2CO3 (pH 11) were incubated with 50 μg of divinylsulfonyl (DVS)-activated Sepharose [100 μg Sepharose beads, 10 μl DVS in 100 μl of 0.5 m K2CO3 (pH 11), 70 min, RT under stirring] and the mixture was evenly distributed into two Mobicol microcolumns. Glycoprobe coupling was carried out overnight at RT under continuous shaking (800 rpm), then the microcolumn was washed with 50 mm NH4OAc (pH 4) and 0.1 m Tris (pH 8) and reequilibrated with either CM or NCM depending on the experiment to conduct. For each of the four glycotopes, two microcolumns for replicate performance were prepared. Additional microcolumns with no immobilized glycan, serving as blanks, were prepared by activating Sepharose as above with monofunctional MVS instead of DVS, then washed and equilibrated with either CM or NCM as above. In a typical experiment, ∼1.5 × 106 entire sperm cells were loaded immediately after swim-up treatment (with either CM or NCM) on the microcolumns and incubated for 24 h at 37 °C with agitation by combined vibration/rotation in an IntelliMixer apparatus (http://www.elmi-tech.com/rm/). Flow-through from each column, containing unbound sperm, was collected and the column washed with the corresponding medium until only residual spermatozoa were observed by microscopy. Sugar-lectin complexes were then digested overnight with trypsin (150 μg/ml) in 25 mm NH4HCO3, pH 7.8, 37 °C, with stirring. After digestion, each column flow-through, containing nonspecific digestion products, was removed and columns washed again with culture media. After gently washing until no spermatozoa were observed, specific-bound peptides were eluted (2 × 300 μl ACN-H2O (2:1) 0.1% TFA, 15 min, 37 °C, stirring). In excision experiments with Sia-containing (3′-SLN and SLex) microcolumns, an additional, competitive elution was done with 400 μl of 0.5 mm fetuin (Sigma-Aldrich, Madrid, Spain) for 15 min, 37 °C, with stirring. Fetuin was removed by filtration (Amicon 30 kDa) and all elution samples were lyophilized prior to LC-MS/MS analysis. In total, for each pair of microcolumns prepared for every glycoprobe, three replicates (for each CM and NCM conditions) were performed, i.e. six replicates per glycoprobe, plus six blank replicates (three replicates per each CM and NCM blank columns). Thus, a total of 30 CREDEX-MS excision experiments (24 with glycoprobe + 6 with blank microcolumns) were carried out per each sperm condition. Microcolumn functionality was tested by running binding tests with specific, pure lectins before excision experiments, as well as in between replicates and at the end of each replication set. Briefly, 20 μg of lectin (UEA-I for Fuc4N; LTA for Lea; MAA for 3′-SLN and AAL for SLex) were added to the corresponding glycoprobe-Sepharose microcolumn and incubated in 100 μl HEPES running buffer (10 mm HEPES, 150 mm NaCl, 5 mm CaCl2 and 1 mm MnCl2, pH 7.4) for 24 h at 37 °C. Unbound material was removed by extensive washing with running buffer, then bound lectin was eluted with 0.1% TFA in 2:1 (v/v) ACN:H2O, except for Sia-containing columns (3′-SLN and SLex glycoprobes), for which a second competitive elution with 1 mm fetuin was performed. The protein contents of each fraction (flow through, wash and elutions) were analyzed by 1D-SDS-PAGE electrophoresis, and preservation of column functionality was confirmed by detection of the specific lectin gel band in the elution fraction. In addition, to further ensure that columns were suitable for reuse, a hydration (HEPES buffer) - dehydration (0.1% TFA in 2:1 (v/v) ACN/H2O) washing cycle was carried out after each excision experiment replicate, to eliminate excess BSA from the CM or to remove residual fetuin used in elution from Sia-containing microcolumns. Proteomic analyses were performed on the elution fractions from all CREDEX experiments and with sperm trypsinization samples (including all the replicates). In order to evaluate instrumental reproducibility for each blank biological replicate, three analyses (three analytical replicates) were performed reaching a total of 18 blank replicates per each capacitating condition. In total, 54 sample injections (12 fetuin glycoprobe elutions, 24 standard glycoprobe elutions and 18 blank elutions) per each sperm condition (CM and NCM) were performed for MS/MS analysis of CREDEX experiments. In order to minimize instrumental variability, a defined batch file was programmed for sample injection. Specifically, samples were injected in groups of nine including the six replicates of the same glycotope alternated with three sample blanks. Additionally, six sample injections of the triplicates of sperm trypsinization experiments per condition were also injected separately. In order to improve the assignments in the subsequent MS/MS analysis, prior to injection, lyophilized samples containing tryptic peptides were resuspended in 200 mm NH4HCO3, reduced with DTT (60 nmol, 1 h, 37 °C), alkylated in the dark with iodoacetamide (120 nmol, 30 min, 25 °C) and purified in UltraMicroSpin C18 columns (The Nest Group, Inc, Southborough, MA). Desalted and purified peptides were dried in a vacuum centrifuge and redissolved in H2O (0.1% HCOOH) for subsequent MS analysis. Samples were analyzed in an LTQ-Orbitrap Velos Pro instrument (Thermo Fisher Scientific, San Jose, CA) coupled to an EasyLC (Thermo Fisher Scientific (Proxeon), Odense, Denmark). Peptides were loaded at 1.5–2 μl/min directly onto a reverse-phase column (12 cm × 75 μm, C18, 3 μm; Nikkyo Technos Co., Ltd. Japan), washed with 4–5 times the injection volume and separated by linear gradients of 3–7% B in A over 1 min (A: 0.1% HCOOH in H2O; B: 0.1% HCOOH in MeCN), followed by 7–35% B in A over 40 min, at a flow rate of 300 nL/min. After each analysis, the column was washed with 90% B for 10 min. The mass spectrometer was operated in positive ionization mode with nanospray voltage set at 2.2 kV and source temperature at 325 °C. Ultramark 1621 (Thermo) was used for external mass analyzer calibration prior to analyses. Moreover, internal calibration was also performed using the background polysiloxane signal at m/z 445.1200. The instrument was operated in data dependent acquisition (DDA) mode and full MS scans at resolution of 60,000 FWMH were used over a mass range of m/z 30–2000. Auto gain control (AGC) was set to 1 × 106, dynamic exclusion (60 s) and charge state filtering disqualifying singly charged peptides was activated. In each DDA cycle, after each survey scan the top ten most intense multiply charged ions above a threshold count of 10,000 were selected for fragmentation at a normalized collision energy of 35%. Fragment ion spectra produced via high-energy collision dissociation (HCD) were acquired in the Orbitrap mass analyzer at a resolution of 7,500, with AGC set at 5 × 104, an isolation window of 2.0 m/z, activation time of 0.1 ms and maximum injection time of 100 ms. All data were acquired and processed with the Xcalibur software v2.2. Proteome Discoverer software (v1.4, Thermo Fisher Scientific) and the Mascot search engine (v2.3.1, Matrix Science) were used for peptide identification and quantification. Searches using the UniProtKB/Swiss-Prot manually annotated and reviewed database were preferred over other alternatives (e.g. TrEMBL), to ensure high quality annotations and avoid redundancy. Specifically, data were searched against an in-house-generated database containing all (6,121) UniProtKB/Swiss-Prot Bos taurus proteins plus common contaminants (∼600 entries). A precursor ion mass tolerance of 7 ppm at the MS1 level was used, and up to three missed cleavages for trypsin were allowed. Fragment ion mass tolerance was set to 20 mmu. Met oxidation, N-terminal acetylation, and Ser, Thr and Tyr phosphorylation were defined as variable modifications and Cys carbamidomethylation as a fixed modification. False discovery rate (FDR) in peptide identification was evaluated by using a decoy database and was set to a maximum of 5%. The log2 corresponding to the average area of the three most intense peptides per protein as calculated by Proteome Discoverer was used as quantitation indicator. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (28Vizcaino J.A. Deutsch E.W. Wang R. Csordas A. Reisinger F. Rios D. Dianes J.A. Sun Z. Farrah T. Bandeira N. Binz P.A. Xenarios I. Eisenacher M. Mayer G. Gatto L. Campos A. Chalkley R.J. Kraus H.J. Albar J.P. Martinez-Bartolome S. Apweiler R. Omenn G.S. Martens L. Jones A.R. Hermjakob H. ProteomeXchange provides globally coordinated proteomics data submission and dissemination.Nat. Biotechnol. 2014; 32: 223-226Crossref PubMed Scopus (2071) Google Scholar) (http://www.proteomexchange.org/) via the PRIDE partner repository with the data set identifier PXD003386. Script tasks and protein list comparisons were performed using the R software (http://www.R-project.org) and the gplots, v.2.11.3 package was used for plotting data in a graphical matrix. A 1/0 matrix for presence/absence analysis minimizing the risk of false positives or negatives was generated by defining changes in protein identification as relevant if occurring in two thirds (67%) of total replicates. Subsequent hierarchical clustering was performed following a Euclidean distance metric and maximum linkage criteria, and final heat maps/dendrograms were generated by displaying x- (sample clustering) versus y-axis (clustering of identified proteins) data. Proportioned Venn diagrams were drawn using Venn Diagram Plotter (http://omics.pnl.gov/software/VennDiagramPlotter.php). Proteins identified were matched against a database containing all published studies on bovine sperm proteomics. PubMed (http://www.ncbi.nlm.nih.gov/pubmed/) and UniProt Knowledgebase (http://www.uniprot.org) were also used, whenever needed, to analyze identified proteins, especially to check which had been previously described in bovine sperm. For comparisons, Swiss-Prot accession number (if available) or protein names (in which case all alternative names were verified) were used. Gene Ontology (GO) resources and tools available at agriGO (a GO analysis toolkit and database for the agricultural community) were used to obtain all existing GO annotations available for k
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