Structural Characterization of Native Mouse Zona Pellucida Proteins Using Mass Spectrometry
2003; Elsevier BV; Volume: 278; Issue: 36 Linguagem: Inglês
10.1074/jbc.m304026200
ISSN1083-351X
AutoresEmily S. Boja, Tanya Hoodbhoy, Henry M. Fales, Jurrien Dean,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoThe zona pellucida is an extracellular matrix consisting of three glycoproteins that surrounds mammalian eggs and mediates fertilization. The primary structures of mouse ZP1, ZP2, and ZP3 have been deduced from cDNA. Each has a predicted signal peptide and a transmembrane domain from which an ectodomain must be released. All three zona proteins undergo extensive co- and post-translational modifications important for secretion and assembly of the zona matrix. In this report, native zonae pellucidae were isolated and structural features of individual zona proteins within the mixture were determined by high resolution electrospray mass spectrometry. Complete coverage of the primary structure of native ZP3, 96% of ZP2, and 56% of ZP1, the least abundant zona protein, was obtained. Partial disulfide bond assignments were made for each zona protein, and the size of the processed, native protein was determined. The N termini of ZP1 and ZP3, but not ZP2, were blocked by cyclization of glutamine to pyroglutamate. The C termini of ZP1, ZP2, and ZP3 lie upstream of a dibasic motif, which is part of, but distinct from, a proprotein convertase cleavage site. The zona proteins are highly glycosylated and 4/4 potential N-linkage sites on ZP1, 6/6 on ZP2, and 5/6 on ZP3 are occupied. Potential O-linked carbohydrate sites are more ubiquitous, but less utilized. The zona pellucida is an extracellular matrix consisting of three glycoproteins that surrounds mammalian eggs and mediates fertilization. The primary structures of mouse ZP1, ZP2, and ZP3 have been deduced from cDNA. Each has a predicted signal peptide and a transmembrane domain from which an ectodomain must be released. All three zona proteins undergo extensive co- and post-translational modifications important for secretion and assembly of the zona matrix. In this report, native zonae pellucidae were isolated and structural features of individual zona proteins within the mixture were determined by high resolution electrospray mass spectrometry. Complete coverage of the primary structure of native ZP3, 96% of ZP2, and 56% of ZP1, the least abundant zona protein, was obtained. Partial disulfide bond assignments were made for each zona protein, and the size of the processed, native protein was determined. The N termini of ZP1 and ZP3, but not ZP2, were blocked by cyclization of glutamine to pyroglutamate. The C termini of ZP1, ZP2, and ZP3 lie upstream of a dibasic motif, which is part of, but distinct from, a proprotein convertase cleavage site. The zona proteins are highly glycosylated and 4/4 potential N-linkage sites on ZP1, 6/6 on ZP2, and 5/6 on ZP3 are occupied. Potential O-linked carbohydrate sites are more ubiquitous, but less utilized. The zona pellucida is an extracellular matrix surrounding mammalian eggs that functions in taxon-specific gamete binding, provides a post-fertilization block to polyspermy, and protects the developing pre-implantation embryo (1Talbot P. Shur B.D. Myles D.G. Biol. Reprod. 2003; 68: 1-9Crossref PubMed Scopus (175) Google Scholar, 2Evans J.P. Florman H.M. Nat. Cell Biol. 2002; 4: S57-S63Crossref PubMed Google Scholar, 3Herrler A. Beier H.M. Cells Tissues. Organs. 2000; 166: 233-246Crossref PubMed Scopus (46) Google Scholar). The mouse zona pellucida (ZP) 1The abbreviations used are: ZP, zona pellucida; CID, collision-induced dissociation; IAA, iodoacetamide; 4-VP, 4-vinylpyridine; TCEP, tris(2-carboxyethyl)phosphine hydrochloride; PNGase F, peptide N-glycosidase F; Gal, galactose; GalNAc, N-acetylgalactosamine; HAc, acetic acid; MS, mass spectrometry.1The abbreviations used are: ZP, zona pellucida; CID, collision-induced dissociation; IAA, iodoacetamide; 4-VP, 4-vinylpyridine; TCEP, tris(2-carboxyethyl)phosphine hydrochloride; PNGase F, peptide N-glycosidase F; Gal, galactose; GalNAc, N-acetylgalactosamine; HAc, acetic acid; MS, mass spectrometry. is composed of three major glycoproteins (ZP1, ZP2, and ZP3) that are synthesized and secreted by oocytes during a 2–3 week growth period (4Wassarman P.M. Annu. Rev. Biochem. 1988; 57: 415-442Crossref PubMed Scopus (475) Google Scholar). The primary structures of ZP1 (623 amino acids), ZP2 (713 amino acids), and ZP3 (424 amino acids) have been deduced from cDNA (5Ringuette M.J. Chamberlin M.E. Baur A.W. Sobieski D.A. Dean J. Dev. Biol. 1988; 127: 287-295Crossref PubMed Scopus (139) Google Scholar, 6Liang L.-F. Chamow S.M. Dean J. Mol. Cell. Biol. 1990; 10: 1507-1515Crossref PubMed Scopus (148) Google Scholar, 7Epifano O. Liang L.-F. Familari M. Moos Jr., M.C. Dean J. Development. 1995; 121: 1947-1956PubMed Google Scholar). Each glycoprotein has a signal peptide directing it into a secretory pathway, a ∼260 amino acid zona domain containing 8 conserved cysteine residues, and a transmembrane domain near the C terminus followed by a short cytoplasmic tail (8Rankin T. Dean J. Rev. Reprod. 2000; 5: 114-121Crossref PubMed Scopus (53) Google Scholar). The zona domain has been observed in multiple proteins (9Bork P. Sander C. FEBS Lett. 1992; 300: 237-240Crossref PubMed Scopus (287) Google Scholar) and has been implicated in the polymerization of extracellular matrices (10Jovine L. Qi H. Williams Z. Litscher E. Wassarman P.M. Nat. Cell Biol. 2002; 4: 457-461Crossref PubMed Scopus (262) Google Scholar). During oocyte growth, ZP1, ZP2, and ZP3 traffick through the growing oocyte, and their ectodomains are released from a transmembrane domain at the surface of the cell (11Rankin T. Familari M. Lee E. Ginsberg A.M. Dwyer N. Blanchette-Mackie J. Drago J. Westphal H. Dean J. Development. 1996; 122: 2903-2910PubMed Google Scholar, 12Zhao M. Gold L. Ginsberg A.M. Liang L.-F. Dean J. Mol. Cell. Biol. 2002; 22: 3111-3120Crossref PubMed Scopus (37) Google Scholar). A conserved hydrophobic patch upstream of the transmembrane domain is required for progression to the cell surface 2M. Zhao, unpublished observations.2M. Zhao, unpublished observations. and a consensus cleavage site (RX(K/R)R↓) for the proprotein convertase furin is present upstream of the transmembrane domain. Although this site has been implicated in the release of the zona ectodomain (13Yurewicz E.C. Hibler D. Fontenot G.K. Sacco A.G. Harris J. Biochim. Biophys. Acta. 1993; 1174: 211-214Crossref PubMed Scopus (79) Google Scholar, 14Litscher E.S. Qi H. Wassarman P.M. Biochemistry. 1999; 38: 12280-12287Crossref PubMed Scopus (66) Google Scholar, 15Williams Z. Wassarman P.M. Biochemistry. 2001; 40: 929-937Crossref PubMed Scopus (49) Google Scholar), mutations (RNRR→ ANAA, or RNRR→ ANGE), do not prevent incorporation of reporter-ZP3 proteins into the zona pellucida in growing oocytes (12Zhao M. Gold L. Ginsberg A.M. Liang L.-F. Dean J. Mol. Cell. Biol. 2002; 22: 3111-3120Crossref PubMed Scopus (37) Google Scholar, 16Qi H. Williams Z. Wassarman P.M. Mol. Biol. Cell. 2002; 13: 530-541Crossref PubMed Scopus (68) Google Scholar) or transgenic mice (12Zhao M. Gold L. Ginsberg A.M. Liang L.-F. Dean J. Mol. Cell. Biol. 2002; 22: 3111-3120Crossref PubMed Scopus (37) Google Scholar) and secretion of recombinant human ZP3 with a similar mutation (RNRR→ ANAA) is not prevented (17Kiefer S.M. Saling P. Biol. Reprod. 2002; 66: 407-414Crossref PubMed Scopus (36) Google Scholar). The three zona proteins are extensively co- and post-translationally modified and a detailed structural analysis of mouse zona pellucida glycans has been reported (18Easton R.L. Patankar M.S. Lattanzio F.A. Leaven T.H. Morris H.R. Clark G.F. Dell A. J. Biol. Chem. 2000; 275: 7731-7742Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). These observations are of particular interest because of the proposal that sperm bind to ZP3 O-glycans linked to Ser332 and Ser334, and the corollary that their removal by glycosidases released from egg cortical granules prevent sperm binding after fertilization (19Wassarman P.M. Mt. Sinai J. Med. 2002; 69: 148-155PubMed Google Scholar). However, there has been controversy as to the nature of the glycans involved and the candidacy of individual terminal sugars as sperm receptors has not been supported by targeted null mutations in mice (8Rankin T. Dean J. Rev. Reprod. 2000; 5: 114-121Crossref PubMed Scopus (53) Google Scholar, 18Easton R.L. Patankar M.S. Lattanzio F.A. Leaven T.H. Morris H.R. Clark G.F. Dell A. J. Biol. Chem. 2000; 275: 7731-7742Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Moreover, recent genetic studies suggest that sperm binding to the zona pellucida is predicated on the three-dimensional structure of the zona pellucida matrix rather than a specific carbohydrate side chain. Cleavage of ZP2 by a protease released during cortical granule exocytosis that occurs upon fertilization may be sufficient to modify the supramolecular structure of the zona matrix and render it non-permissive to sperm binding (20Rankin T.L. Coleman J.S. Epifano O. Hoodbhoy T. Turner S.G. Castle P.E. Lee E. Gore-Langton R. Dean J. Dev. Cell. 2003; 5: 33-43Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Many of these controversies stem from the paucity of biological material that makes robust biochemical analysis difficult and has prompted reliance on recombinant zona proteins expressed in heterologous systems where processing and modifications may differ from those in mouse oocytes. This report takes advantage of microscale LC-MS to partially characterize mouse ZP1, ZP2, and ZP3 as a mixture in native zonae pellucidae. A hybrid QTOF instrument has the advantages of high mass accuracy, great sensitivity and resolution, and is well suited for detection of low levels of biological materials. Using these technologies we have determined both N and C termini, intramolecular disulfide linkages, and have identified N- and O-glycosylation sites on mouse ZP1, ZP2, and ZP3. Materials—Urea, dithiothreitol, iodoacetamide (IAA), 4-vinylpyridine (4-VP), and ammonium bicarbonate were purchased from Sigma-Aldrich Co. Tris[2-carboxyethyl]phosphine hydrochloride (TCEP, 0.5 m) was obtained from Pierce Biotechnology, Inc. (Rockford, IL). Sequencing grade porcine trypsin was from Promega, Inc. (Madison, WI) and Asp-N was from Roche Applied Science (Indianapolis, IN). All HPLC solvents were of the highest grade commercially available from J. T. Baker (Philipsburg, NJ). Glycopro Deglycosylation Kit was obtained from Prozyme Inc. (San Leandro, CA). An anti-rat secondary IgG-conjugated to horseradish peroxidase was obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). All NOVEX gels were obtained from Invitrogen (Carlsbad, CA). Deglycosylation and Proteolytic Digestion—Zonae pellucidae were isolated from an ovarian homogenate using density gradient ultracentrifugation (21Bleil J.D. Wassarman P.M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6778-6782Crossref PubMed Scopus (294) Google Scholar). Approximately 20 μg of zona proteins were lyophilized prior to denaturation in 4 μl of 8 m urea in 250 mm Tris-HCl, pH 8.0 at 37 °C for 1 h. Reduction with dithiothreitol (5 mm final concentration) and subsequent alkylation with IAA (80 mm final concentration) were performed in the same buffer at 37 °C for 1 h each. To this reaction mixture was added 100 μl of 50 mm ammonium bicarbonate, pH 7.8. The excess reagents including urea, dithiothreitol, and IAA were removed by buffer exchange (3×) using an YM-10 Amicon centrifugation filter device with a MW cutoff of 10 kDa (Millipore Corp., Bedford, MA). The proteins were re-dissolved in 50 μl of 50 mm ammonium bicarbonate, pH 7.8 and deglycosylated using a Prozyme Glycopro Deglycosylation Kit. N-glycans were removed using 1 μl of PNGase F (5000 units/ml) for 26 h at 37 °C. After N-deglycosylation, the sample was divided into two fractions and lyophilized. Half of the material was reconstituted in 50 mm ammonium bicarbonate buffer, pH 6.1, prior to O-glycan removal. O-Deglycosylation was performed using 1 μl of the following exoglycosidases: sialidase A (5 units/ml), β-(1–4)-galactosidase (3 units/ml), and β-N-acetylglucosaminidase (45 units/ml) ± 1 μl of endo-O-glycosidase (1.25 units/ml) at 37 °C for 36 h. The pH of this sample was raised to 6.5 in the middle of the reaction. The O-deglycosylated samples were subsequently lyophilized, and re-dissolved in 50 mm ammonium bicarbonate buffer, pH 7.8, to give ∼10 pmol/μl final concentration of ZP3 in the ZP mix. 1 μl of ZP mix (containing 10 pmol of ZP3) was digested in a 10-μl volume consisting of 1 μl of acetonitrile, 7 μl of 50 mm ammonium bicarbonate buffer, pH 7.8, and either 1 μl of trypsin (1 pmol) for 18 h, Asp-N (0.5 pmol) for 18 h, or trypsin (1 pmol) for 48 h followed by Asp-N (0.5 pmol) for an additional 18 h. Trypsin cleaves C-terminal to lysine and arginine; Asp-N cleaves N-terminal to aspartic acid, although infrequent cleavage N-terminal to glutamic acid also has been reported (22Lopaticki S. Morrow C.J. Gorman J.J. J. Mass Spectrom. 1998; 33: 950-960Crossref PubMed Google Scholar). Disulfide Linkage Mapping—A non-reduced zona protein mixture (20 μg) was denatured in 8 m urea, pH 7.2 at 37 °C for 1 h. Free thiols of cysteine residues were blocked with 1 m (final concentration) of 4-VP in a 25-μl reaction mixture prepared in an ammonium bicarbonate buffer, pH 7.2 containing 10% methanol (23Sechi S. Chait B.T. Anal. Chem. 1998; 70: 5150-5158Crossref PubMed Scopus (354) Google Scholar). The excess reagents were removed as described above, and the pH of the solution was brought to 7.5 prior to N-deglycosylation with PNGase F and proteolytic digestions. Throughout the entire experiment, the reaction pH was carefully controlled in the range of 7.2–7.5 to preserve native disulfide linkages. Disulfide bonds were determined by analyzing the proteolytic fragments using LC-MS. To confirm these linkages, TCEP (0.5–1 mm final concentration) was added to reduce the pre-existing disulfide-bonded peptides at 37 °C for 1 h. LC-MS Analysis of Protein Digests—Trypsin, Asp-N, and trypsin/Asp-N double digests of ZP mix were analyzed on a Micromass QTOF Ultima Global (Micromass, Manchester, UK) in electrospray mode interfaced with an Agilent HP1100 CapLC (Agilent Technologies, Palo Alto, CA) prior to the mass spectrometer. 2 μl (∼2 pmol) of each digest was loaded onto a Vydec C18 MS column (100 × 0.15 mm; Grace Vydec, Hesperia, CA) and chromatographic separation was performed at 1 μl/min using the following gradient: 0–10% B over 5 min; gradient from 10–40% B over 60 min; 40–95% B over 5 min; 95% B held over 5 min (solvent A: 0.2% formic acid in water; solvent B: 0.2% formic acid in acetonitrile). A data-dependent analysis (DDA) method collected CID data for the three most abundant peptide ions observed in the preceding survey scan (m/z 300–1990) above a threshold of 10 counts/sec. Collision energy for CID experiments was optimized using peptide standards with a wide mass range (m/z 400–1600) and charge state (+1 to +4) and was typically between 20–65 eV. Data was processed using the MassLynx software package (version 3.5) to generate peak list files before submitting them to in-house licensed Mascot search (24Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6661) Google Scholar) (biospec.nih.gov (MatrixScience Ltd., London, UK)). Error tolerant searches were performed to consider irregular cleavages and post-translational modifications. In addition, manual data analysis in search of specific ions of interest was carried out. All MS/MS fragment ions were within 50 ppm of their theoretical values determined by the BioLynx Protein/Peptide Editor and most were within 10 ppm. Gel Electrophoresis and Western blotting—Zona proteins were solubilized in 2× denaturing and reducing Laemmli sample buffer (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar) and separated by one-dimensional SDS-PAGE on a 4–20% NOVEX Trisglycine gel at 120 V. The proteins were then electroblotted onto a NitroPure-supported nitrocellulose membrane (45-μm pore diameter; OSMONICS INC., Westborough, MA) at 25 V for 1 h. Nonspecific binding was blocked by incubating the nitrocellulose in phosphate-buffered saline containing 0.1% Tween-20 and 10% nonfat dried milk for1hat room temperature. Proteins were immunoblotted overnight at 4 °C in the same blocking solution containing one of the following rat monoclonal antibodies specific to: ZP1 (m1.4, 1:100 hybridoma supernatant) (26Rankin T.L. Tong Z.-B. Castle P.E. Lee E. Gore-Langton R. Nelson L.M. Dean J. Development. 1998; 125: 2415-2424PubMed Google Scholar), ZP2 (IE3, 1:100 hybridoma supernatant) (27East I.J. Dean J. J. Cell Biol. 1984; 98: 795-800Crossref PubMed Scopus (55) Google Scholar), and ZP3 (IE10, 1:1000 IgG fraction isolated from hybridoma supernatant) (28East I.J. Gulyas B.J. Dean J. Dev. Biol. 1985; 109: 268-273Crossref PubMed Scopus (83) Google Scholar). The blots were washed three times (15 min each) with phosphate-buffered saline containing 0.1% Tween-20, and then incubated in an anti-rat secondary IgG-conjugated to horseradish peroxidase for 1 h at room temperature. Immunoblotted bands were washed again and then visualized by enhanced chemiluminescence (ECL) according to the manufacturer's instructions (Amersham Biosciences). Preliminary Analysis of the Zona Pellucida—Mass spectrometric analyses were performed on native zonae pellucidae isolated from 500 NIH Swiss mice and purified by density gradient centrifugation. Monoclonal antibodies that recognize peptide epitopes detected mouse ZP1 (average molecular mass, 132 kDa), ZP2 (120 kDa) and ZP3 (79 kDa) on immunoblots after samples had been reduced and alkylated (data not shown). Following treatment with PNGase F to remove N-linked glycans, there was a dramatic shift in the apparent molecular mass of ZP1 (132 kDa → 105 kDa), ZP2 (120 kDa → 68 kDa) and ZP3 (79 kDa → 44 kDa), similar to those reported earlier for ZP2 and ZP3 (29Nagdas S.K. Araki Y. Chayko C.A. Orgebin-Crist M.-C. Tulsiani D.R.P. Biol. Reprod. 1994; 51: 262-272Crossref PubMed Scopus (60) Google Scholar). Additional treatment with a mixture of exo- and endo-O-glycosidase resulted in a less diffuse band for ZP1 and ZP3 and a further shift in average molecular masses to 63 and 39 kDa, respectively. However, there was no apparent shift in the molecular mass of ZP2, confirming previous observations (29Nagdas S.K. Araki Y. Chayko C.A. Orgebin-Crist M.-C. Tulsiani D.R.P. Biol. Reprod. 1994; 51: 262-272Crossref PubMed Scopus (60) Google Scholar). Although glycoproteins run anomalously on SDS-PAGE (30Leach B.S. Collawn Jr., J.F. Fish W.W. Biochemistry. 1980; 19: 5734-5741Crossref PubMed Scopus (193) Google Scholar), these results suggest that ZP1 is more heavily O- than N-glycosylated, ZP2 is predominantly N-glycosylated with little or no O-glycosylation, and ZP3 is predominantly N-glycosylated with relatively little O-glycosylation. Each sample analyzed by mass spectrometry was a mixture of zona proteins with ZP2 and ZP3 present in approximately equal amounts and ZP1 much less abundant (31Bleil J.D. Wassarman P.M. Dev. Biol. 1980; 76: 185-202Crossref PubMed Scopus (458) Google Scholar). Using a combination of proteolytic enzymes before and after enzymatic deglycosylation, 56% of the polypeptide chain of mature ZP1 (see Supplemental Table IA), 96% of mature ZP2 (see Supplemental Table IB) and 100% of mature ZP3 (see Supplemental Table IC) was identified by mass spectrometry. Although looked for, two or more ions ascribable to other known proteins were not observed in the zona preparation with the exception of clusterin/apolipoprotein J/sulfated glycoprotein 2 from Mus musculus (32French L.E. Chonn A. Ducrest D. Baumann B. Belin D. Wohlwend A. Kiss J.Z. Sappino A.P. Tschopp J. Schifferli J.A. J. Cell Biol. 1993; 122: 1119-1130Crossref PubMed Scopus (110) Google Scholar). This protein, implicated in cell-cell adhesions of epithelia tissues including the early embryo, was identified by CID spectra of two peptides 385VSTVTTHSSDSEVPSR400 and 401VTEVVVK407. Whether clusterin participates in the zona pellucida matrix or its presence reflects a minor contamination of the zona preparation remains to be determined. Determination of the N Termini of ZP1, ZP2, and ZP3— Virtually all extracellular proteins have N-terminal signal peptides that direct them into secretory pathways and are removed in the endoplasmic reticulum by signal peptidases. A predictive algorithm (33Von Heijne G. Nucleic Acids Res. 1986; 14: 4683-4690Crossref PubMed Scopus (3685) Google Scholar) predicts cleavage of ZP1, ZP2 and ZP3 immediately upstream of Gln21, Val35, and Gln23, respectively. Edman degradation sequence confirmed the N terminus of ZP2 (6Liang L.-F. Chamow S.M. Dean J. Mol. Cell. Biol. 1990; 10: 1507-1515Crossref PubMed Scopus (148) Google Scholar), but was either imprecise for ZP1 (7Epifano O. Liang L.-F. Familari M. Moos Jr., M.C. Dean J. Development. 1995; 121: 1947-1956PubMed Google Scholar) or uninformative for ZP3 (5Ringuette M.J. Chamberlin M.E. Baur A.W. Sobieski D.A. Dean J. Dev. Biol. 1988; 127: 287-295Crossref PubMed Scopus (139) Google Scholar). Peptide mapping of ZP1 from Asp-N digestion followed by LC-MS indicated that the N terminus starts at Gln21, which had been converted to pyroglutamate. The CID spectrum (Fig. 1A) of the precursor ion at m/z 811.372+ (inset, calc. 811.392+) corresponding to the mass of the N-terminal peptide 21qRLHLEPGFEYSY33 (q = pyroglutamate) indicated the presence of both y and b ion series including y1–2, y2-H2O, y7, b2–6, b8–10, b2-NH3, b4-NH3, b6-NH3. In addition, an ion series a5–6, a9, and a11 as well as immonium ions of tyrosine and phenylalanine were observed. MS data from the combined trypsin/Asp-N digestion revealed the presence of the [M+2H]2+ ion at m/z 915.45 (inset, calc. 915.462+) corresponding to the N-terminal carbamidomethylated peptide 35VSLPQSENPAFPGTLIC51 of ZP2 (Fig. 1B). The CID spectrum of this ion generated many internal fragment ions (PG, PQ, PGT, PGTLI, PQSENPAF, etc.) near proline residues and, together with sequence ions y1, y2, y6, and a4, b2-H2O, b7-NH3, b11, confirmed its identity. For mouse ZP3, tryptic digestion revealed [M+3H]3+ and [M+4H]4+ at m/z 702.42 and 527.06 that match the N-terminal peptide 23qTLWLLPGGTPTPVGSSSPVK43, again with a pyroglutamate in place of a glutamine (Fig. 1C). Unfortunately, the low abundance of these multiply charged ions prevented them from being selected for fragmentation (CID). Furthermore, the highly charged state of this peptide is unusual since there is only one basic lysine residue. However, gas phase basicity can promote proton trapping by proline, tryptophan, and glutamine (34Smith R.D. Loo R.A. Loo R.R. Busman M. Udseth H.R. Mass. Spec. Rev. 1991; 10: 359-451Crossref Scopus (599) Google Scholar, 35Schnier P.D. Gross D.S. Williams E.R. J. Am. Soc. Chem. 1995; 117: 6747-6757Crossref Scopus (207) Google Scholar) and may account for these observations. Determination of the C Termini of ZP1, ZP2, ZP3—A potential proprotein convertase (furin) cleavage site (RX(R/K)R↓) that lies 35–40 amino acids N-terminal of the transmembrane domain is conserved among the mouse zona proteins and has been implicated in the release of the mature zona ectodomain (13Yurewicz E.C. Hibler D. Fontenot G.K. Sacco A.G. Harris J. Biochim. Biophys. Acta. 1993; 1174: 211-214Crossref PubMed Scopus (79) Google Scholar). Because trypsin cuts within the furin site and could have provided ambiguous results, samples were digested with Asp-N. MS data was obtained from both N-deglycosylated and N/O-deglycosylated zonae pellucidae. For mouse ZP1, we observed a peptide of MH+ 774.42 Da corresponding to the sequence of 540DSGIARR546 both as a +1 (calc. 774.421+) and +2 charged ion at m/z 387.72 (Fig. 2A). This indicates that the C terminus of mouse ZP1 (Arg546) lies two amino acids upstream of the furin cleavage site. Due to the low abundance of these ions, CID data were not obtained. For ZP2, Asp-N digestion and LC-MS data revealed the presence of a precursor ion of MH+ 1649.76 representing the C-terminal peptide 619DSPLCSVTCPASLRS633 where Cys623 and Cys627 were both carbamidomethylated (calc. MH+ 1649.76). The CID spectrum of the +2 charged ion of this peptide at m/z 825.38 confirmed the identity of the peptide through the b ion series of peptide fragments (b2, b3-H2O, b4, b4-H2O), as well as the y ion series (y6-y12, y6-NH3, y9-NH3, y10-H2O) (Fig. 2B). Hence, the C terminus of ZP2 (Ser633) also lies two amino acids upstream of the furin cleavage site. ZP3, in which there was no convenient aspartate residue, was digested with PNGase F, which released protein-bound N-glycans and converted Asn330 to aspartic acid. Subsequent Asp-N digestion and LC-MS revealed the presence of the C-terminal peptide 330DSSSSQFQIHGPRQWSKLVSRN351 (Fig. 2C), and its identity was confirmed by CID (y3-y6, y122+ y132+, y152+, y162+ as well as a2, b2, b2-H2O, b3-H2O, b4-H2O). Thus, the C terminus of ZP3 lies at Asn351. Taken together, these mass spectrometric data indicated that the primary cleavage site of native ZP1, ZP2, and ZP3 lies N-terminal to a dibasic motif that is part of, but distinct from, the proprotein convertase (furin) cleavage site. Disulfide Linkage Mapping—Blocking with 4-VP at pH 7.2 revealed no S-pyridylethylated cysteine-containing peptides in the mixture, suggesting that all cysteines (at least those detected in the digest) participate in disulfide bonding. In the following discussion, the two disulfide bonded peptide chains have been arbitrarily designated as P1 and P2, priming fragmentations that arise from the latter, e.g. y′. Because the disulfide bridge is sometimes "reductively" cleaved either between or on each side of sulfur, peptide fragment ions will appear carrying either an SH or SSH at the cysteine site, and these are referred to as yr (or y′r) and yd (or y′d), respectively. ZP1 forms a homodimer in the native zona pellucida. It has 21 cysteine residues and the potential to form 10 intramolecular disulfide bonds with the remaining cysteine residue available for intermolecular ZP1-ZP1 linkage. However, due to the low abundance of ZP1 in the zona protein mixture only one disulfide-bonded peptide was detected. The low abundances of the +3 and +4 charged ions at m/z 1351.05 and 1013.50 observed after trypsin digestion arose from 438TDPSLVLLLHQCWATPTTSPFEQPQWPILSDGCPFK473 intramolecularly disulfide-bonded between Cys449 and Cys470 (Fig. 3A and Table I). No CID spectra were obtained, and as expected, both ions disappeared after treatment with tris(2-carboxyethyl)phosphine hydrochloride (TCEP) for 1 h. Unfortunately, the reduced ion 2 Da higher was not available to corroborate the reduction.Table IDisulfide bond linkage mapping of native mouse zona proteinsTable IDisulfide bond linkage mapping of native mouse zona proteins ZP2 has 20 cysteine residues capable of 10 disulfide bonds. Within the zona domain (containing ten cysteines, eight of which are conserved) four out of five possible disulfide bonds were identified (Table I). These linkages were confirmed by observing the disappearance of disulfide-bridged ions described below upon TCEP treatment and/or by sequence obtained from CID. Cys365/Cys457 formed a disulfide pair as observed by ions at m/z 696.832+ (calc. 696.802+) and 464.883+ (calc. 464.873+) derived from the trypsin/Asp-N digest (data not shown). The calculated MH+ of the S-S linked peptides 362DELCAQ367 (P1) and 457CYYIR462 (P2) is 1392.59 Da, which is in good agreement with our experimental values. The CID spectrum of 464.883+ generated partial sequence ions of y1–2 and b2 from P1, as well as y1′ and immonium ion of tyrosine residues from P2 (data not shown). The Cys396/Cys417 disulfide pair in ZP2 was observed by a very low abundance +4 charged ion at m/z 836.41 (MH+ 3342.72). This ion derived from trypsin digestion corresponds to the peptides 382PALNLDTLLVGNSSCQPIFK401 (Asn to Asp conversion at position 393 after PNGase F treatment) joined with 410FHIPLNGCGTR420 via a S-S bond (combined masses of two peptides minus 2 Da). Although the CID spectrum of this ion was unavailable, 836.414+ disappeared after TCEP reduction. Furthermore, two ions showed up at m/z 1066.052+ and 607.812+ that correspond to 382PALNLNTLLVGNSSCQPIFK401 (Asn393 → Asp393) and 410FHIPLNGCGTR420 in their reduced state. This observation adds confidence in the assignment of this disulfide linkage even without CID data. Two more disulfide links in ZP2 provided +3 and +4 charged ions at m/z 1198.59 and 899.19 (MH+ 3593.73), which correspond to the intramolecularly disulfide-bonded peptide 599GLSSLIYFHCSALICNQVSLDSPLCSVTCPASLR632 formed between the four cysteines within the same tryptic peptide (2 disulfide bonds with a loss of 4 Da). The CID spectrum of 1198.933+ did not generate many sequence ions as expected from its size and the two internal cystine linkages. Thus, the actual disulfide pairing among these four cysteines was indeterminate from trypsin digestion alone. However, this problem was resolved when additional Asp-N cleavage revealed the presence of the peptide 619DSPLCSVTCPASLR632 linked via Cys623/Cys627, as detected by ions at m/z 723.872+ and traces of 482.923+. This linkage was corroborated by the disappearance of the ion at m/z 723.872+ after TCEP reduction, and the appearance of an ion at m/z 724.812+ corresponding to the above peptide with its free sulfhydryl groups. Thus, the second disulfide linkage must join Cys608 and Cys613. A disulfide bond between Cys84 and Cys102, near the N terminus of ZP2, outside the zona domain was also identified. The +3, +4, and +5 charged ions at m/z 1269.95, 952.71, and 762.35 (MH+ 3807.87) with strong ion intensities correspond to the accurate mass of the S-S-linked peptides 69WNPSVVDTLGSEILNCTYALDLER92 (P1; Asn83 → Asp83 conversion) and 97F
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