corresponding author: dr. emily s. boja laboratory of ...structural characterization of native mouse...

Structural Characterization of Native Mouse Zona Pellucida Proteins Using Mass Spectrometry

Emily S. Boja*†1, Tanya Hoodbhoy§1, Henry M. Fales*, Jurrien Dean§

*Laboratory of Biophysical Chemistry, NHLBI, §Laboratory of Cellular Developmental Biology, NIDDK, National Institutes of Health,

Bethesda, MD 20892

Running title: Mass Spectrometric Characterization of Mouse Zona Pellucida Proteins †Corresponding Author: Dr. Emily S. Boja Laboratory of Biophysical Chemistry, NHLBI 50 South Drive Room 3122, Building 50 Bethesda, MD 20892-8014 Tel: (301) 496-5628 Fax: (301) 402-3404 Email: [email protected]

JBC Papers in Press. Published on June 10, 2003 as Manuscript M304026200 by guest on M

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SUMMARY 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

amino termini of ZP1 and ZP3, but not ZP2, were blocked by cyclization of glutamine to

pyroglutamate. The carboxyl 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.

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INTRODUCTION

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 (1-3). The mouse

zona pellucida (ZP2) is composed of three major glycoproteins (ZP1, ZP2 and ZP3) that

are synthesized and secreted by oocytes during a two-three week growth period (4).

The primary structures of ZP1 (623 amino acids), ZP2 (713 amino acids) and ZP3 (424

amino acids) have been deduced from cDNA (5-7). 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

carboxyl terminus followed by a short cytoplasmic tail (8). The ‘zona domain’ has been

observed in multiple proteins (9) and has been implicated in the polymerization of

extracellular matrices (10).

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

(11;12). A conserved hydrophobic patch upstream of the transmembrane domain is

required for progression to the cell surface (unpublished observations) and a consensus

cleavage site (RXK/RR↓ ) 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 (13-15), mutations (RNRR→ANAA, or RNRR→ANGE), do not prevent

incorporation of reporter-ZP3 proteins into the zona pellucida in growing oocytes (12;16)


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or transgenic mice (12) and secretion of recombinant human ZP3 with a similar

mutation (RNRR→ANAA) is not prevented (17).

The three zona proteins are extensively co- and post-translationally modified and a

detailed structural analysis of mouse zona pellucida glycans has been reported (18).

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 (19). 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 (8;18). 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 (20).

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


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and resolution, and is well suited for detection of low levels of biological materials. Using

these technologies we have determined both amino and carboxyl termini, intramolecular

disulfide linkages and have identified N- and O-glycosylation sites on mouse ZP1, ZP2

and ZP3.

EXPERIMENTAL PROCEDURES

Materials. Urea, dithiothreitol (DTT), iodoacetamide (IAA), 4-vinylpyridine (4-VP) and

ammonium bicarbonate were purchased from Sigma-Aldrich Co. (St. Louis, MO). 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 Diagnostics, Inc.

(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 (21). 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 37oC for 1 h. Reduction with DTT (5 mM final concentration) and

subsequent alkylation with IAA (80 mM final concentration) were performed in the same

buffer at 37oC for 1 h each. To this reaction mixture was added 100 µl of 50 mM


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ammonium bicarbonate, pH 7.8. The excess reagents including urea, DTT and IAA

were removed by buffer exchange (3X) using an YM-10 Amicon centrifugation filter

device with a MW cutoff of 10 kD (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 37oC. 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 37oC 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

approximately 10 pmol/µl final concentration of ZP3 in the ZP mix. One 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 (22).

Disulfide Linkage Mapping. A non-reduced zona protein mixture (20 µg) was denatured

in 8 M urea, pH 7.2 at 37oC for 1 h. Free thiols of cysteine residues were blocked with 1


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M (final concentration) of 4-VP in 25 µl reaction mixture prepared in an ammonium

bicarbonate buffer, pH 7.2 containing 10% methanol (23). 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 37oC

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. Two µl (approximately 2

pmol) of each digest was loaded onto a Vydec C18 MS column (100 x 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


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processed using the MassLynx software package (version 3.5) to generate peak list

files before submitting them to in-house licensed Mascot search (24)

(http://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 2X

denaturing and reducing Laemmli sample buffer (25) and separated by one-dimensional

SDS-PAGE on a 4-20% NOVEX Tris-Glycine gel at 120V. 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. Non-specific binding

was blocked by incubating the nitrocellulose in PBS containing 0.1% Tween-20 and

10% nonfat dried milk for 1 h at room temperature. Proteins were immunoblotted

overnight at 40C in the same blocking solution containing one of the following rat

monoclonal antibodies specific to: ZP1 (m1.4, 1:100 hybridoma supernatant)(26), ZP2

(IE3, 1:100 hybridoma supernatant)(27), and ZP3 (IE10, 1:1000 IgG fraction isolated

from hybridoma supernatant)(28). The blots were washed three times (15 min each)

with PBS 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, Piscataway, NJ).


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RESULTS

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 kD), ZP2 (120 kD) and ZP3 (79 kD) 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 kD → 105 kD), ZP2 (120 kD → 68 kD) and ZP3 (79 kD → 44 kD), similar to those

reported earlier for ZP2 and ZP3 (29). 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 kD and 39 kD, respectively. However, there was no

apparent shift in the molecular mass of ZP2, confirming previous observations (29).

Although glycoproteins run anomalously on SDS-PAGE (30), 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 (31).

Using a combination of proteolytic enzymes before and after enzymatic deglycosylation,

56% of the polypeptide chain of mature ZP1 (Supplemental Table IA), 96% of mature

ZP2 (Supplemental Table IB) and 100% of mature ZP3 (Supplemental Table IC) was


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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 (32). This protein,

implicated in cell-cell adhesions of epithelia tissues including the early embryo, was

identified by CID spectra of two peptides (385)VSTVTTHSSDSEVPSR(400) and

(401)VTEVVVK(407). 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 Amino 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 (33) predicts cleavage of ZP1, ZP2 and ZP3

immediately upstream of Gln21, Val35 and Gln23, respectively. Edman degradation

sequence confirmed the N-terminus of ZP2 (6), but was either imprecise for ZP1 (7) or

uninformative for ZP3 (5).

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 (21)qRLHLEPGFEYSY(33)

(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


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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 (35)VSLPQSENPAFPGTLIC(51) 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 (23)qTLWLLPGGTPTPVGSSSPVK(43), 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 (34;35) and may account for these

observations.

Determination of the Carboxyl Termini of ZP1, ZP2, ZP3

A potential proprotein convertase (furin) cleavage site (RXR/KR↓ ) 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 (13).

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


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and N/O-deglycosylated zonae pellucidae. For mouse ZP1, we observed a peptide of

MH+ 774.42 Da corresponding to the sequence of (540)DSGIARR(546) both as a +1 (calc.

774.421+) and +2 charged ion at m/z 387.72 (Fig. 2A). This indicates that the carboxyl

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 (619)DSPLCSVTCPASLRS(633) 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 carboxyl

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 (330)DSSSSQFQIHGPRQWSKLVSRN(351) (Fig 2C) and its identity was

confirmed by CID (y3-y6, y122+, y13

2+, y152+, y16

2+ 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.


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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 S, 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

(438)TDPSLVLLLHQCWATPTTSPFEQPQWPILSDGCPFK(473) intramolecularly disulfide-

bonded between Cys449 and Cys470 (Fig. 3A, 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.


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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 (362)DELCAQ(367) (P1) and (457)CYYIR(462) (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 (382)PALNLDTLLVGNSSCQPIFK(401) (Asn to Asp

conversion at position 393 after PNGase F treatment) joined with

(410)FHIPLNGCGTR(420) 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 (382)PALNLNTLLVGNSSCQPIFK(401) (Asn393 →Asp393) and

(410)FHIPLNGCGTR(420) in their reduced state. This observation adds confidence in the

assignment of this disulfide linkage even without CID data.


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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 (599)GLSSLIYFHCSALICNQVSLDSPLCSVTCPASLR(632) 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 (619)DSPLCSVTCPASLR(632) 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 (69)WNPSVVDTLGSEILNCTYALDLER(92) (P1;

Asn83→Asp83 conversion) and (97)FPYETCTIK(105) (P2). Moreover, the CID spectra of

both 952.954+ and 1270.303+ showed a similar fragmentation pattern corresponding to

the sequence of both peptides linked via disulfides (Fig. 3B). The presence of y1-8 ions

of P1 from the ion at m/z 952.954+ showed fragmentation up to Cys84 where the


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disulfide linkage was located. The linkage was confirmed by analysis of the combined

trypsin and Asp-N cleavage (data not shown). The ions at m/z 892.442+ and 595.293+

correspond to the mass of the disulfide-linked peptide (83)NCTYAL(88) (Asn83→Asp83)

and (97)FPYETCTIK(105) (MH+ 1783.8). These ions disappeared upon reduction with

TCEP and additional ions corresponding to their reduced forms at m/z 685.26+1

[(83)NCTYAL(88)] (Asn83→Asp83) and 551.252+[(97)FPYETCTIK(105)] were generated,

further confirming the original disulfide linkage between the two peptides.

The mature mouse ZP3 amino acid sequence is essentially a compact ‘zona domain’.

There are 12 cysteines in the mature form with four of them clustered near the C-

terminus outside the ‘zona domain’ (Table I). In the first pair, masses corresponding to

the peptide (44)VECLEAELVVTVSR(57) disulfide-linked to (133)VEVPIECR(140) (with loss of

2 Da) were observed at m/z 622.834+ and 830.123+ from both the trypsin only and Asp-

N/trypsin double digest (data not shown). These ions, however, were not selected for

fragmentation by the software. After reduction, these ions vanished while ions at m/z

773.912+ and 472.752+ corresponding to both reduced peptides respectively were

detected. In the second pair, a precursor ion of MH+ 3058.45 as detected by its +3 and

+4 charged ions at m/z 1020.16 and 765.37 corresponds to

(65)LVQPGDLTLGSEGCQPR(81) (P1) disulfide-bridged to (91)FNAQLHECSSR(101) (P2).

The CID spectrum of m/z 765.62+4 yielded a y ion series including y1-3 ions prior to and

y5r past Cys78 of P1, as well as the b2-4 ions (Fig. 3C). In addition, P2 generated (y’1-3)

ions, followed by y7’r past Cys98, and sequential b ions including (b’2-5), b7’ and b8

’r. This

disulfide linkage was further confirmed by the results from Asp-N/ trypsin double


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digestion. The ions at m/z 855.393+ and 641.784+ correspond to the mass of two

peptides linked via a S-S bond (MH+ 2564.16) in the same sequence region:

(70)DLTLGSEGCQPR(81) and (91)FNAQLHECSSR(101).

Cys216/Cys283 within the ‘zona domain’ of ZP3 formed a disulfide bridge as shown by

the ions at m/z 780.353+ and 585.524+. These ions (MH+ 2339.08) derived from a

trypsin/Asp-N digest represent the disulfide-linked peptides (214)DHCVATPSPLP(224) (P1)

and (277)NTLYITCHLK(286) (P2). Although CID data were not available, reduction with

TCEP produced two ions at m/z 568.742+ and 603.302+ corresponding to the individual

peptides with free sulfhydryl groups, confirming the original disulfide bridge. Traces of

(277)NTLYITCHLK(286) with a free sulfhydryl group (unmodified by 4-VP) were also

detected under non-reducing conditions. This observation could result from disulfide

displacement of the peptide (277)NTLYITCHLK(286) (originally linked to Cys216) by the –

SH group of a cysteine residue from other sources.

Since six out of a total of eight cysteines in mZP3 had been accounted for in disulfide

bonding, it seemed reasonable that the last linkage would be between

(236)DFHGCLV(242) and (300)ACSF(303). However, ions corresponding to this linkage

calculated as MH+ 1214.50 Da were not detected in the double digest sample. A +1

charged ion at m/z 427.16 corresponding to the mass of (300)ACSF(303) was detected

only after TCEP reduction, but not present in the non-reduced digest. Similarly, the +1

and +2 charged ions at m/z 790.31 and 395.66 which correspond to (236)DFHGCLV(242)


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were only detected after TCEP treatment. This observation implies that Cys301 was

originally linked to Cys240.

Lastly, the C-terminal peptide (306)TSQSWLPVEGDADICDCCSHGNCSNSSSS

QFQIHGPR(342) with two asparagines at positions 327 and 330 converted to aspartic

acids (see below) was internally disulfide-bonded twice, as demonstrated by the

presence of a +4 charged ion at m/z 988.41 (MH+ 3950.68). The conversion of two

asparagines to aspartic acids resulted in a mass increase of 1.97 Da, however, the loss

of 4.03 Da from formation of two disulfide bridges caused a net decrease of 2.06 Da (or

0.51 Da for a +4 charged ion). The CID spectrum of 988.904+ identified the y ion series

including y1-7, y4-NH3, y7-NH3, y9, y12, together with b6-H2O and some internal fragments

such as PV and HG (data not shown). The immonium ions of glutamine, histidine and

tryptophan residues at m/z 101.07, 110.07 and 159.09 were also observed, although

direct evidence of the exact cystine bridging among this group of four cysteines could

not be determined. The disappearance of 988.414+ together with the detection of

989.404+ (MH+ 3954.61) after TCEP reduction further confirmed this disulfide linkage.

N-linked Glycosylation Sites

N-glycosylation of proteins occurs only at asparagine residues within the consensus

sequence NXS/T where X cannot be a proline. PNGase F endoglycosidase releases

protein-bound N-linked glycans and by converting the involved asparagine residue to an

aspartic acid provides a signature increase in mass (0.98 Da). There are four predicted

N-linked glycosylation sites that follow the NXS/T sequence motif in native secreted ZP1


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and six in both ZP2 and ZP3. In ZP1, all four predicted asparagines at positions 49, 68,

240 and 371 were N-glycosylated within the mature protein (Table IIA). Figure 4A

provides an example of the CID spectrum of a +3 charged Cys-carbamidomethylated

peptide (368)CIFNASDFLPIQASIFSPQPPAPVTQSGPLR(398) at m/z 1119.53

(MH+=3356.57) derived from trypsin digestion. The MH+ ion of this peptide is 0.87 Da

higher than the expected value (MH+=3355.70), suggesting that Asn371 was converted

to Asp. Fragmentation generated a series of b ions (b2-b12 and b14-b16), as well as y ion

series including y4-y5, y7, y9-y16, y13-H2O, y14-NH3, y92+, y12

2+, y142+, y15

2+, y162+, y22

2+,

y232+ confirming the peptide sequence. The b4-9, b11-12 and b14-16 ions clearly

demonstrated a change of Asn to Asp at position 371 upon PNGase F treatment. In

order to obtain more sequence information, additional proteolytic cleavages were

subsequently carried out.

In ZP2, all six N-glycosylation sites were occupied (Table IIB). Trypsin digestion after

PNGase F treatment clearly showed that four Asn residues at positions 83, 172, 184

and 393 were converted to Asp. In Figure 4B, glycosylation site identification by CID is

illustrated for the +3 charged ion of a glycopeptide

(69)WNPSVVDTLGSEILNCTYALDLER(92) at m/z 922.74 derived from trypsin digestion.

Again, the experimental precursor ion MH+ 2766.20 is 0.86 Da higher than the

calculated value of this peptide (MH+ 2765.34). The y10-15 ions unequivocally confirm the

conversion of Asn to Asp at position 83 within the N-glycosylation motif [(83)NCT85)]. The

presence of y1, y2-H2O, y3-NH3, y3-9, b2, b5-NH3/H2O, b4-6, b8-9, b12-13 and a14 ions further

confirm the sequence identity and demonstrates that Asn70 preceding a proline was not


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N-glycosylated, as predicted. In addition, the +2 charged ion of this peptide at m/z

1383.65 was observed (0.95 Da higher than the calculated mass) and its CID spectrum

showed a very similar fragmentation pattern to that of the +3 charged ion (data not

shown).

As a result of Asp-N as well as trypsin/Asp-N sequential digestion, Asn217 and Asn264

were also identified as N-glycosylation sites in ZP2. In the case of Asn264, a peptide

(264)NATHMTLTIPEFPGK(278) resulting from the double digest was detected at m/z

829.41 (+2 charged) and 553.27 (+3 charged), a mass increase of 0.98 Da which was

further confirmed by the CID spectrum of 829.412+ (data not shown). This conversion

led to Asp-N cleavage at position 264 which allowed detection of this peptide. The same

observation was made with a peptide

(217)NATGIVHYVQESSYLYTVQLELLFSTTGQK(246) at m/z 1130.89 (+3 charged)

derived from the sequential digest that resulted from the Asn-Asp conversion at position

217 for Asp-N cleavage. In both cases, a mass increase of 0.93-0.98 Da was noted

from the conversion.

Similarly, trypsin digestion of ZP3 generated five out of six Asp-containing peptides after

PNGase F deglycosylation (Table IIC). A +4 charged ion at m/z 1046.43 indicates that

the C-terminal peptide (306)TSQSWLPVEGDADICDCCSHGNCSNSSSSQFQIHGPR(342)

was N-glycosylated both Asn327 and Asn330. Interestingly the observation of a second

co-eluting ion at m/z 1046.164+ implies the presence of another population of the same

peptide N-glycosylated at either Asn327 or Asn330 (data not show). However, no CID


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data was available to locate the precise glycosylated site on the second ion. The very

large tryptic peptide fragment from residue 185 to 256 encompassing the predicted N-

glycosylation site Asn227 was not detected. To obtain additional information on this

middle region of the ZP3 sequence, Asp-N as well as trypsin/Asp-N sequential digestion

was performed. Two Asp-N fragments, (214)DHCVATPSPLPDPNSSPYHFIV(235) and

(225)DPNSSPYHFIV(235) at m/z 817.373+ and 638.292+, the masses of which match the

calculated values of these peptides (MH+ 2450.14 and 1275.60) clearly indicated the

absence of a predicted N-linked Asn227 residue. Asn304 was found to be N-

glycosylated from the tryptic peptide (300)ACSFNK(305) showing a mass shift of +0.98 Da

and confirmed by the CID spectrum of its +2 charged ion at m/z 364.16 (data not

shown). Further confirmation for this N-linked asparagine site came from Asp-N

digestion where a peptide (295)DKLNKACSF(303) was observed at m/z 541.762+ due to

the generation of a new cleavage site at position 304 after PNGase F deglycosylation.

The same observation was made with a trypsin/Asp-N fragment

(330)NSSSSQFQIHGPR(342) at m/z 723.342+ and 482.563+ where Asp-N cleavage

occurred at Asn330 indicated a mass shift of +0.95 Da. As shown in Figure 4C, low

energy CID generated the sequential y ion series y1-y10 and y6-NH3 ions, as well as b2-

b3 and b2-H2O ions. Hence, the N-terminal Asn330 of this ZP3 peptide was

unambiguously assigned as an N-glycosylation site.

O-linked Glycosylation Sites

Although O-glycans attach to threonines and serines, there is no specific consensus

sequence to readily predict potential linkage sites. Instead, monosaccharides must be


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removed by a series of exoglycosidases (sialidase A, β(1-4) galactosidase, β-N-

acetylglucosaminidase) until only the Galβ(1-3)GalNAc core remains attached to the

serine/threonine residues. This results in a mass increase of 365.13 Da/core glycan

over the basic peptide. Further O-deglycosylation with endo-O-glycosidase removes the

core sugar leaving serine and threonine residues unmodified. Shifts in mobility on SDS-

PAGE after deglycosylation suggest that ZP1 contains considerably more O-linked

carbohydrate side chains than either ZP2 or ZP3 (data not shown)(29), although

estimates of glycosylation based on SDS-PAGE are inexact (30). However, due to its

low abundance, no mass spectrometric data was obtained on ZP1 O-linked

glycosylation. Based on the near complete coverage of ZP2 prior to enzymatic removal

of O-linked carbohydrates (96%), there appears to be only one potential O-linkage site

(T455). The absence of a significant shift in apparent molecular mass in SDS-PAGE

after enzymatic removal of O-linked glycans, suggests that few, if any, serine/threonine

residues are occupied or are at low occupancy below our detection limit (data not

shown) (29).

Two ZP3 domains were identified that contain one or more O-linked oligosaccharide

side chains: one at the N-terminus (residues 23-43 with 5 potential sites) and the other

within the ‘zona domain’ (residues 144-168 with six potential sites). The concomitant

identification of peptides from these domains prior to deglycosylation implies a mixture

of ZP3 molecules, some with O-glycans and others without. Multiply charged ions (+3

and +4) at m/z 702.42 and 527.06 of the N-terminal ZP3 peptide

(23)qTLWLLPGGTPTPVGSSSPVK(43) (where q is a pyroglutamate) were detected in the


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N/O-deglycosylated sample (Fig. 1C), but not in the N-deglycosylated sample. Although

these masses do not contain the Galβ(1-3)GalNAc mass shift, this observation raised

the possibility that O-glycosylation at these predicted sites may initially be present and

that the labile O-linked carbohydrate groups were lost during MS analysis. In addition,

the presence of a +3 charged ion at m/z 1067.47 corresponding to this N-terminal

peptide with the attachment of 3 Galβ(1-3)GalNAc moieties (i.e., with a mass increase

of 3 x 365.13 Da) in the N/O-deglycosylated sample supports O-glycosylation at three of

these potential sites (data not shown). No CID spectrum of this ion was obtained due to

its low abundance. This +3 charged ion, however, disappeared upon endo-O-

glycosidase treatment. Differences (10 min) in chromatographic elution suggests that

both glycosylated and unglycosylated species are present in the native zona pellucida.

Thus, three among the five potential sites (Thr32, Thr34, Ser38, Ser39, Ser40) appear

to have O-linked glycans and only Thr32, Thr34, Ser39 are predicted to be glycosylated

(probabilities of 79%, 76%, 72%,

respectively)(http://www.cbs.dtu.dk/services/NetOGlyc/)(36).

In the N/O-deglycosylated sample, the +2 and +3 charged ions of the unglycosylated

ZP3 peptide (144)QGNVSSHPIQPTWVPFR(160) were detected at m/z 976.0 and 650.98

respectively (a mass increase of 0.98 as a result of the Asn-Asp conversion at position

146). The CID spectrum of m/z 976.02+ confirmed the sequence by the presence of y1-5,

y7-11 ions, as well as b2-5, b7 and b9 ions. The b3-5, b7, b9 ions demonstrated that Asn146

was converted to an aspartic acid upon PNGase F deglycosylation (Fig. 5A). This


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suggests that a population of this peptide was either not O-glycosylated, or the labile

sugar core structure was lost during MS analysis. The presence of a Galβ(1-3)GalNAc

core was detected by the ions at m/z 1158.562+ and 772.713+ (corresponding to a mass

increase of 365.13 Da). Selected ion chromatograms for ions at m/z 976.02+

(unglycosylated) and m/z 772.713+ (glycosylated) co-eluted during chromatography

which is uncommon for differentially glycosylated species and is more consistent with

the labile sugar core structure being lost during MS analysis. The CID spectrum of

773.033+ showed the presence of not only sequence ions resulting from the peptide

backbone including b2-3, b2-H2O, b3-H2O, as well as y1, y3-4, y7-8, y10-13, y10-H2O, y11-NH3

ions, but also low mass carbohydrate marker ions at m/z 204.09 (GalNAc+H+), 168.08

[(GalNAc-2H2O)+H+], 144.08 [(GalNAc-HAc)+H+] and 366.14 [(Galβ(1-3)GalNAc)+H+]

(Fig. 5B). Moreover, these ions were no longer present upon deglycosylation with

endoglycosidases, again supporting that this peptide was previously O-glycosylated

(data not shown). Unfortunately, even with CID data, we could not determine the exact

site of the sugar linkage among the three potential sites (Ser148, Ser149, Thr155) due

to the loss of the sugar moiety prior to the peptide backbone cleavage. However,

Thr155 is a predicted O-linked glycosylation site in mouse ZP3 with a probability of 98%

(http://www.cbs.dtu.dk/services/NetOGlyc/).

Similarly, a +2 charged ion at m/z 608.27 eluting early in the chromatogram from the

tryptic digest of the N/O-deglycosylated sample corresponds to the mass of the ZP3

peptide (161)ATVSSEEK(168) with the Galβ(1-3)GalNAc core attached, presumably to


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either Thr162 or one of the two serines at positions 164 and 165. The CID spectrum of

this ion produced only MH+-[Gal(β1-3)GalNAc] at m/z 850.41, perhaps due to being

subjected to CID late in peak elution when less precursor ion signal is available.

However, its low mass carbohydrate marker ions including GalNAc+H+, (GalNAc-

H2O)+H+, (GalNAc-2H2O)+H+, and (GalNAc-HAc)+H+, at m/z 204.09, 186.08, 168.08

and 144.07 resembled that of the O-glycosylated peptide described above, indicating

that this peptide is clearly O-glycosylated. The lack of peptide ions with sugar moieties

attached made it impossible to assign the site of the O-glycan linkage, but based on the

predictive algorithm, Thr162 has a 70% probability of being glycosylated.

Earlier studies have described mouse ZP3 as the primary sperm receptor, an activity

ascribed to O-glycans attached at Ser332 and Ser334 (37;38). However, the

trypsin/Asp-N digest of the native ZP mixture generated the masses at m/z 723.342+

and 482.563+ as described above (Fig. 4C). These masses correspond to the peptide

(330)DSSSSQFQIHGPR(342), where Asp-N cleavage took place at Asn330 due to the

Asn-Asp conversion (i.e., a mass shift of +0.95 Da). Since these masses match the

calculated masses of this peptide with the replacement of Asn with Asp (MH+ 1445.68)

without any prior O-deglycosylation treatment (N-deglycosylated sample), and since the

peptide identity was confirmed by CID sequence data, it indicates that neither Ser332

nor Ser334 are O-glycosylated at a measurable level. Because glycosylation at these

sites was inferred from previous mutational studies (37), we looked specifically for the

masses corresponding to various combinations of glycosylation sites using extracted ion

chromatograms in the N/O-deglycosylated samples, but did not find them. Thus, to the


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extent of our mass spectrometric detection (low femtomole levels), we did not observe

glycosylation of any potential O-glycosylation sites except an N-terminal cluster

(predicted to be Thr32, Thr34, Ser39) and a second cluster in the ‘zona domain’

(predicted to be Thr155, Thr162).

DISCUSSION

The mammalian zona pellucida is a unique biological structure that surrounds growing

oocytes, ovulated eggs and the pre-implantation embryo (39). Although essential for in

vivo fertilization and early development, its biochemical characterization has been

impeded by the difficulty of purifying adequate quantities of native material. Earlier

studies had determined the presence of three major glycoproteins (ZP1, ZP2, ZP3) and

their primary structures have been deduced from cDNA (8). More recent genetic studies

using null mutations and replacement with human homologues have provided insight

into the molecular basis of sperm binding to the zona matrix (20;26). We now report the

biochemical analysis of ZP1, ZP2 and ZP3 in native mouse zonae pellucidae without

further purification of individual proteins. Taking advantage of highly accurate and

sensitive mass spectrometry, structural features of individual mouse zona pellucida

proteins including N- and C-termini, presence of intramolecular disulfide linkages and

sites of N- and O-glycosylation have been determined.

Proteolytic Processing of Zona Pellucida Proteins

The three zona proteins are distinct from one another with ZP1 and ZP2 more

evolutionarily conserved than ZP3 (40). However, as a cohort they share certain


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common features. Each has a signal peptide to direct it into a secretory pathway and

each has an ectodomain that must be released from a transmembrane domain prior to

incorporation into the extracellular zona matrix. The native N-terminus of each zona

protein was determined by mass spectrometry. Both ZP1 (Fig. 6) and ZP3 (Fig. 8) are

blocked by a pyroglutamate (pyroGln21 and pyroGln23, respectively) and the N-terminal

Val35 of ZP2 (Fig. 7) confirms an earlier determination by Edman degradation (6). Thus,

the signal peptides of ZP1, ZP2 and ZP3 are 20, 34 and 22 amino acids long,

respectively, and the experimentally determined cleavage sites correspond to those of

von Hejine’s predictive algorithm (33).

Once directed into the secretory pathway, the zona proteins remain associated with the

endomembrane system until they are released at the surface of the oocytes. There has

been controversy as to the cleavage site required for release of the ectodomain from

the predicted transmembrane domain near the carboxyl terminus (12;14;15;17). The

mass spectrometric data indicates that the C-termini of ZP1 (Arg546), ZP2 (Ser633) and

ZP3 (Asn351) in native zonae pellucidae are N-terminal to a dibasic motif (ZP1, Arg547-

Arg548; ZP2, Lys634-Arg635; ZP3, Arg352-Arg353). These presumed cleavage sites

are part of, but distinct from, a proprotein convertase (furin) site (13) that is imperfectly

conserved among zona proteins. The ZP1, ZP2 and ZP3 dibasic motif lies 43, 50, 37

amino acids, respectively, upstream of the mouse protein transmembrane domains and

is conserved in all mammalian species examined to date. It has been suggested that

similarly positioned C-termini in the quail and Xenopus homologues of ZP3 result from

cleavage at the proprotein convertase followed by carboxylpeptidase trimming of two


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basic residues (41;42). The observation that mutation of the dibasic motif does not

preclude secretion and incorporation of mouse ZP3 into the zona pellucida suggests

that alternative cleavage sites are available as has been reported for other secreted

proteins (43;44).

Thus, after N- and C-terminal processing, the polypeptide chains of ZP1 (Fig. 6), ZP2

(Fig. 7) and ZP3 (Fig. 8) will have molecular masses of 58kD, 68kD and 36kD,

respectively. These predictions are in good agreement with the apparent molecular

masses observed after N/O-deglycosylation of ZP1 (63kD), ZP2 (68kD) and ZP3 (39kD)

in native zonae pellucidae by immunoblot (data not shown) and autoradiography (29).

The minor discrepancies may reflect residual O-linked sugars predicted after enzymatic

deglycosylation or aberrant migration and are well within estimation errors associated

with SDS-PAGE.

Formation of Intramolecular Disulfide Bonds within the ‘Zona Domain’

Disulfide linkages are thought to be one of the major factors in stabilizing native

conformations of secreted proteins (45;46). No free cysteine residues were detected in

the native zona pellucida proteins and intermolecular disulfide bonds have been

observed only in ZP1 (31). A ~260 amino acid ‘zona domain’ with eight conserved

cysteine residues is present in ZP1 (amino acids 288-542), ZP2 (amino acids 363-630)

and ZP3 (amino acids 45-308)(9). The mass spectrometric data is most complete for

the mouse ZP3 ‘zona domain’ in which four disulfide bonds are defined (Fig. 8). The two

N-terminal bonds (Cys46/Cys139; Cys78/Cys98) form 1-4 and 2-3 linkages (loop-within-


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loop) and the two C-terminal disulfide bonds (Cys216/Cys283; Cys240/Cys301) form 1-

3 and 2-4 crossover linkages. The four additional cysteine residues in ZP3 (Cys320,

Cys322, Cys323, Cys328) lie C-terminal to the ‘zona domain’ and form two disulfide

bonds, the linkage of which is indeterminate due to their tight clustering within nine

amino acid residues.

Although incompletely determined, the formation of disulfide bonds in the ‘zona domain’

of ZP1 (Fig. 6) and ZP2 (Fig. 7) appear to differ from that of ZP3. The two, N-terminal

bonds (Cys365/Cys457; Cys396/Cys417) in the ZP2 ‘zona domain’ conform with the

loop within a loop motif observed in ZP3, but the two disulfide bonds at the C-terminus

of the ZP2 ‘zona domain’ (Cys608/Cys613); Cys623/Cys627) do not share the ZP3

cross-over motif. Disulfide linkage between the remaining cysteine residues (Cys538,

Cys559) in ZP2 ‘zona domain’ was not determined, but the corresponding residues

(Cys449, Cys470) in ZP1 form a disulfide bond. Thus, there appear to be two additional

residues (beyond the 8 conserved cysteines) in the ‘zona domain’ of ZP1 and ZP2 that

are not present in ZP3 and disulfide bond formations in the C-terminal half of the ZP2

(and perhaps ZP1) zona domain differ from those of ZP3.

The ‘zona domain’ has been implicated in forming protein polymers not only in the zona

pellucida matrix, but between constituents of the extracellular tectorin membrane found

in the inner ear (10;47). Genetically altered mice lacking ZP1 form a zona matrix

composed of ZP2 and ZP3 (48); mice lacking ZP2 form a thinner, more fragile matrix

composed of ZP1 and ZP3 (49); but mice lacking ZP3 do not form a zona pellucida


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(11;50). Thus, a zona matrix can be formed by either ZP1/ZP3 or ZP2/ZP3 consistent

with the necessity of two types of ‘zona domains:’ one from ZP3 and the other either

from ZP1 or ZP2. Taken together these data suggest that the structure of ZP1 and ZP2

‘zona domains’ may be similar to each other and different from that of ZP3.

Glycosylation of Zona Proteins

N-glycosylation plays an essential role in the folding/trafficking of glycoproteins (51;52),

and can only occur at asparagines that have a consensus NXS/T motif (where X cannot

be a proline). O-glycosylation derivatizes the hydroxyl groups of threonine and serine

residues and, although there is no particular sequence motif dictating whether

glycosylation can take place, flanking amino acids are thought to exert an influence

(53;54). Each of the proteolytically processed mouse zona proteins contains a limited

number of potential N-linkage glycosylation sites (ZP1, 4 sites; ZP2, 6 sites; ZP3, 6

sites), but considerably more potential O-linkage sites (ZP1, 82 sites; ZP2, 84 sites;

ZP3, 58 sites). Zona glycoproteins were either N- or N/O-deglycosylated as described

above to identify glycosylated asparagine, serine and threonine residues.

Deglycosylation with PNGase F releases the entire N-glycan bound to asparagine

residues and by converting the residue to aspartic acid provides an unequivocal mass

spectrometric signature of the glycosylation site. All four potential N-linked sites on ZP1

(Asn49, Asn68, Asn240, Asn371) contain carbohydrate side chains (Fig. 6) and all six

sites on ZP2 (Asn83, Asn172, Asn184, Asn217, Asn264, Asn393) are also occupied

(Fig. 7) in accord with early estimates (55;56). Five of the six potential N-linked sites on


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ZP3 (Asn146, Asn273, Asn304, Asn327, Asn330) have carbohydrate side chains (Fig.

8) which is somewhat more extensive than earlier reports (57). Only Asn227 on ZP3

was experimentally determined by mass spectrometry and CID not to be glycosylated,

perhaps due to inaccessibility or the presence of proline residues immediately upstream

and downstream of the consensus motif. Taken together, these data show that all but

one asparagine residue within the NXS/T consensus motif is N-glycosylated in mature,

native ZP1, ZP2 and ZP3. The molecular masses of N-glycans attached to the mouse

zona pellucida ranges from 1.6-3.8 kD (18) and based on the number of side chains it

appears that ~15-30% of the mass of individual mouse zona proteins is N-linked

carbohydrate side chains.

The composition of O-glycans isolated from native mouse zona pellucida has been

determined by chromatography and mass spectrometry (18). Although association with

individual zona proteins was not reported, O-linked sugars ranged in size from three to

six residues, did not include fucose, and the great majority had core-2 type structures,

Gal(β1-3)GalNAc which provides a useful identification tag. We have reasoned that if a

peptide is detected prior to deglycosylation or in an N-deglycosylated sample, then it is

not O-glycosylated. Conversely, O-glycosylated peptides would only be found after

removal of its O-glycans. Exo-O-glycosidases remove O-linked sugars from zona

proteins leaving a Gal(β1-3)GalNAc core attached to serine/threonine residues. Endo-

O-glycosidase can be used in addition to exo-O-glycosidases to remove the core sugars

with no modification of the serine/threonine residues. Thus, in addition to CID data

detecting the attached sugar, the presence of the Gal(β1-3)GalNAc tag (365.13 Da), on


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the serine/threonine residues before, but not after, treatment with Endo-O-glycosidase

is useful in identifying O-glycan sites. However, in view of the fact that evidence has

been found for loss of at least one type of O-linked sugar (mannose) upon collision in a

triple stage quadrupole (58), one must consider the possibility that similar losses of the

closely related O-linked GalNAc residue may arise from collisional processes in the

source region.

Experimental determination by mass spectrometry of O-linked sites on ZP1 and ZP2

was not successful either due to incomplete coverage (ZP1) or a paucity of O-linked

sugars (ZP2). Greater success was obtained with ZP3. Two clusters of O-linked

glycosylation were detected on native ZP3 (Fig. 8). One, at the N-terminus appears to

contain three occupied amino acid residues (predicted to be Thr32, Thr34, Ser39) and a

second in the middle of the ‘zona domain’ with two O-linkage sites (predicted to be

Thr155, Thr162). The identification of peptides from these regions prior to

deglycosylation suggests that O-glycosylation in some cases is heterogeneous with

some ZP3 molecules containing O-glycans and others not.

The biological functions of glycosylation in zona pellucida proteins remain to be

determined. Treatment with tunicamycin which prevents the addition of N-linked sugars

has been variously reported to inhibit or facilitate the secretion of ZP2 and ZP3 (59;60).

More controversially, mouse ZP3 has been described as the primary receptor for sperm

binding, a biologic activity ascribed to oligosaccharide side chains linked to Ser332 and

Ser334 (19). However, neither serine is occupied by O-linked oligosaccharide side


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chains as evidenced by the presence of (330)DSSSSQFQIHGPR(342) under reducing and

non-reducing conditions (confirmed by MS and CID data) which was detected without

any prior O-deglycosylation. Additionally, transgenic mice expressing mutant ZP3

(Ser332→Gly332; Ser334→Ala334) have normal fertility (61), although the more

definitive assessment of their reproductive fitness in the Zp3 null background has not

been reported. Whether the N-terminal or ‘zona domain’ cluster of O-glycans plays a

role in sperm binding remains to be determined, but it seems unlikely that they act as

the sole ‘sperm receptor’ given the genetically altered mice in which sperm continue to

bind to the zona pellucida despite the cortical granule reaction and the release of

putative glycosidases (20).


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Reference List

1. Talbot, P., Shur, B. D., and Myles, D. G. (2003) Biol. Reprod. 68, 1-9

2. Evans, J. P. and Florman, H. M. (2002) Nat. Cell Biol. 4(S1), S57-S63

3. Herrler, A. and Beier, H. M. (2000) Cells Tissues. Organs 166, 233-246

4. Wassarman, P. M. (1988) Annu. Rev. Biochem. 57, 415-442

5. Ringuette, M. J., Chamberlin, M. E., Baur, A. W., Sobieski, D. A., and Dean, J. (1988) Dev. Biol. 127, 287-295

6. Liang, L.-F., Chamow, S. M., and Dean, J. (1990) Mol. Cell. Biol. 10, 1507-1515

7. Epifano, O., Liang, L.-F., Familari, M., Moos, M. C., Jr., and Dean, J. (1995) Development 121, 1947-1956

8. Rankin, T. and Dean, J. (2000) Rev. Reprod. 5, 114-121

9. Bork, P. and Sander, C. (1992) FEBS Lett. 300, 237-240

10. Jovine, L., Qi, H., Williams, Z., Litscher, E., and Wassarman, P. M. (2002) Nat. Cell Biol. 4, 457-461

11. Rankin, T., Familari, M., Lee, E., Ginsberg, A. M., Dwyer, N., Blanchette-Mackie, J., Drago, J., Westphal, H., and Dean, J. (1996) Development 122, 2903-2910

12. Zhao, M., Gold, L., Ginsberg, A. M., Liang, L.-F., and Dean, J. (2002) Mol. Cell. Biol. 22, 3111-3120

13. Yurewicz, E. C., Hibler, D., Fontenot, G. K., Sacco, A. G., and Harris, J. (1993) Biochim. Biophys. Acta 1174, 211-214

14. Litscher, E. S., Qi, H., and Wassarman, P. M. (1999) Biochemistry 38, 12280-12287

15. Williams, Z. and Wassarman, P. M. (2001) Biochemistry 40, 929-937

16. Qi, H., Williams, Z., and Wassarman, P. M. (2002) Mol. Biol. Cell 13, 530-541

17. Kiefer, S. M. and Saling, P. (2002) Biol. Reprod. 66, 407-414

18. Easton, R. L., Patankar, M. S., Lattanzio, F. A., Leaven, T. H., Morris, H. R., Clark, G. F., and Dell, A. (2000) J. Biol. Chem. 275, 7731-7742

19. Wassarman, P. M. (2002) Mt. Sinai J. Med. 69, 148-155


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

35

20. Rankin, T. L., Coleman, J. S., Epifano, O., Hoodbhoy, T., Turner, S. G., Castle, P. E., Lee, E., Gore-Langton, R., and Dean, J. (2003) Developmental Cell, in press.

21. Bleil, J. D. and Wassarman, P. M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6778-6782

22. Ingrosso, D., Fowler, A. V., Bleibaum, J., and Clarke, S. (1989) Biochem. Biophys. Res. Commun. 162, 1528-1534

23. Sechi, S. and Chait, B. T. (1998) Anal. Chem. 70, 5150-5158

24. Perkins, D. N., Pappin, D. J., Creasy, D. M., and Cottrell, J. S. (1999) Electrophoresis 20, 3551-3567

25. Laemmli, U. K. (1970) Nature 227, 680-685

26. Rankin, T. L., Tong, Z.-B., Castle, P. E., Lee, E., Gore-Langton, R., Nelson, L. M., and Dean, J. (1998) Development 125, 2415-2424

27. East, I. J. and Dean, J. (1984) J. Cell Biol. 98, 795-800

28. East, I. J., Gulyas, B. J., and Dean, J. (1985) Dev. Biol. 109, 268-273

29. Nagdas, S. K., Araki, Y., Chayko, C. A., Orgebin-Crist, M.-C., and Tulsiani, D. R. P. (1994) Biol. Reprod. 51, 262-272

30. Leach, B. S., Collawn, J. F., Jr., and Fish, W. W. (1980) Biochemistry 19, 5734-5741

31. Bleil, J. D. and Wassarman, P. M. (1980) Dev. Biol. 76, 185-202

32. French, L. E., Chonn, A., Ducrest, D., Baumann, B., Belin, D., Wohlwend, A., Kiss, J. Z., Sappino, A. P., Tschopp, J., and Schifferli, J. A. (1993) J. Cell Biol. 122, 1119-1130

33. Von Heijne, G. (1986) Nucleic Acids Res. 14, 4683-4690

34. Smith, R. D., Loo, R. A., Loo, R. R., Busman, M., and Udseth, H. R. (1991) Mass. Spec. Rev. 10, 359-451

35. Schnier, P. D., Gross, D. S., and Williams, E. R. (1995) J. Am. Soc. Chem. 117, 6747-6757

36. Hansen, J. E., Lund, O., Engelbrecht, J., Bohr, H., Nielsen, J. O., and Hansen, J. E. (1995) Biochem. J. 308 ( Pt 3), 801-813

37. Chen, J., Litscher, E. S., and Wassarman, P. M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 6193-6197


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

36

38. Williams, Z., Litscher, E. S., and Wassarman, P. M. (2003) Biochem. Biophys. Res. Commun. 301, 813-818

39. Yanagimachi, R. (1994) Mammalian fertilization. In Knobil, E. and Neil, J., editors. The physiology of reproduction, Raven Press, New York

40. Spargo, S. C. and Hope, R. M. (2003) Biol. Reprod. 68, 358-362

41. Sasanami, T., Pan, J., Doi, Y., Hisada, M., Kohsaka, T., and Toriyama, M. (2002) Eur. J. Biochem. 269, 2223-2231

42. Kubo, H., Matsushita, M., Kotani, M., Kawasaki, H., Saido, T. C., Kawashima, S., Katagiri, C., and Suzuki, A. (1999) Dev. Genet. 25, 123-129

43. Blobel, C. P. (2000) Curr. Opin. Cell Biol. 12, 606-612

44. Schwager, S. L., Chubb, A. J., Woodman, Z. L., Yan, L., Mentele, R., Ehlers, M. R., and Sturrock, E. D. (2001) Biochemistry 40, 15624-15630

45. Zapun, A., Jakob, C. A., Thomas, D. Y., and Bergeron, J. J. (1999) Structure Fold. Des 7, R173-R182

46. Fassio, A. and Sitia, R. (2002) Histochem. Cell Biol. 117, 151-157

47. Legan, P. K., Rau, A., Keen, J. N., and Richardson, G. P. (1997) J. Biol. Chem. 272, 8791-8801

48. Rankin, T., Talbot, P., Lee, E., and Dean, J. (1999) Development 126, 3847-3855

49. Rankin, T. L., O'Brien, M., Lee, E., Wigglesworth, K. E. J. J., and Dean, J. (2001) Development 128, 1119-1126

50. Liu, C., Litscher, E. S., Mortillo, S., Sakai, Y., Kinloch, R. A., Stewart, C. L., and Wassarman, P. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5431-5436

51. Parodi, A. J. (2000) Annu. Rev. Biochem. 69, 69-93

52. Scheiffele, P. and Fullekrug, J. (2000) Essays Biochem. 36, 27-35

53. Nehrke, K., Ten Hagen, K. G., Hagen, F. K., and Tabak, L. A. (1997) Glycobiology 7, 1053-1060

54. Nehrke, K., Hagen, F. K., and Tabak, L. A. (1996) J. Biol. Chem. 271, 7061-7065

55. Greve, J. M., Salzmann, G. S., Roller, R. J., and Wassarman, P. M. (1982) Cell 31, 749-759

56. Noguchi, S. and Nakano, M. (1993) Biochim. Biophys. Acta 1158, 217-226


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

37

57. Salzmann, G. S., Greve, J. M., Roller, R. J., and Wassarman, P. M. (1983) EMBO. J. 2, 1451-1456

58. Dobos, K. M., Swiderek, K., Khoo, K. H., Brennan, P. J., and Belisle, J. T. (1995) Infect. Immun. 63, 2846-2853

59. Shimizu, S., Tsuji, M., and Dean, J. (1983) J. Biol. Chem. 258, 5858-5863

60. Roller, R. J. and Wassarman, P. M. (1983) J. Biol. Chem. 258, 13243-13249

61. Liu, C., Litscher, S., and Wassarman, P. M. (1995) Mol. Biol. Cell 6, 577-585


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FOOTNOTES

1These authors contributed equally to the work.

2Abbreviations: ZP, zona pellucida; CID, collision-induced dissociation; IAA, iodoacetamide, 4-VP; 4-vinylpyridine; DTT, dithiothreitol; TCEP, tris(2-carboxyethyl)phosphine hydrochloride, PNGase F, peptide N-glycosidase F, Gal, galactose, GalNAc, N-acetylgalactosamine, HAc, acetic acid, MS, mass spectrometry.


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ACKNOWLEDGMENTS

We appreciate the many useful discussions with members of our laboratories, the initial

help in the project by Stephanie Gill and the critical reading of the manuscript by Dr.

Douglas Sheeley.


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FIGURE LEGEND

Figure 1. Determination of the amino termini of native mouse zona proteins. Asp-N,

trypsin and both were used to map peptides at the N-termini using microscale LC-MS

analysis. (A). The N-terminus of ZP1 defined by the Asp-N peptide

(21)qRLHLEPGFEYSY(33) with pyroglutamate (q) in place of Gln21 exhibits the +2

charged ion at m/z 811.37 Da. CID spectrum confirms the sequence; (B). CID spectrum

of the +2 charged precursor ion at m/z 915.45 Da corresponding to the amino terminal

peptide (35)VSLPQSENPAFPGTLIC(51) of mouse ZP2 derived from sequential trypsin

and Asp-N cleavage. Many internal fragment ions near prolines were observed together

with partial sequence ions from the peptide; (C). The observed masses at m/z 527.06

(+4 charged) and 702.42 (+3 charged) from tryptic cleavage match the expected value

of the N-terminal peptide (23)qTLWLLPGGTPTPVGSSSPVK(43) with a pyroglutamate in

place of a glutamine.

Figure 2. Determination of the carboxyl termini of native mouse zona proteins. Asp-N

cleavage specific at the N-terminus of an aspartic acid residue followed by LC-MS

analysis identified the C-termini as the amino acid preceding a dibasic peptide motif

upstream of the furin consensus cleavage site in all three cases. (A). The C-terminus of

ZP1 as defined by the +1 and +2 charged ions at m/z 774.42 and 387.72 corresponds

to the peptide (540)DSGIARR(546); (B). CID spectrum of the +2 charged ion at m/z 825.38

corresponds to the carboxyl terminal peptide (619)DSPLCSVTCPASLRS(633) of ZP2; (C).

The carboxyl terminal peptide of ZP3 [(330)DSSSSQFQIHGPRQWSKLVSRN(351)] was

detected at m/z 636.80 (+4 charged) as well as 848.75 (+3 charged), 0.96 Da higher


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than expected, demonstrating that Asn330 was replaced by Asp. The CID spectrum

confirmed the sequence identity of this peptide (see text).

Figure 3. Disulfide bond localization of mouse zona proteins. (A). An intramolecular

disulfide-linked peptide (438)TDPSLVLLLHQCWATPTTSPFEQPQWPILSDGCPFK(473)

from ZP1 derived from trypsin digestion is shown at m/z 1013.504+. This, and a second

ion at 1351.053+ (not shown) correspond to the mass of this peptide minus 2 Da due to

the disulfide bridge: (B). CID of the ion at m/z 952.954+ (confirmed by +3 and +5

charged ions at m/z 1269.95 and 762.35) from ZP2 showing fragment ions correspond

to the sequences (69)WNPSVVDTLGSEILDCTYALDLER(92) (P1) and (97)FPYETCTIK(105)

(P2) disulfide bonded to each other. Many ions are formed from “reductive” processes

and contain either CysSH or CysSSH (see text); (C). The disulfide linkage formed

between (65)LVQPGDLTLGSEGCQPR(81) (P1) and (91)FNAQLHECSSR(101) (P2) of ZP3

was detected by ions at m/z 1020.153+ and 765.374+ (precursor ion of MH+ 3058.45).

The CID spectrum as shown here from the latter ion clearly indicated the fragment ions

derived from both peptides connected via a disulfide bond.

Figure 4. Localization of N-glycosylation sites in mouse zona proteins by mass

spectrometry. (A). ZP1 N-linked glycopeptide (368)CIFDASDFLPIQASIFSP

QPPAPVTQSGPLR(398) at m/z 1119.53+3; (B). ZP2 N-linked glycopeptide

(69)WNPSVVDTLGSEILDCTYALDLER(92) at m/z 922.74+3; (C). ZP3 N-linked

glycopeptide (330)DSSSSQFQIHGPR(342) at m/z 482.56+3 resulting from trypsin/Asp-N

sequential digestion clearly shows that the Asn-Asp conversion at position 330 resulting


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in a mass increase of 0.95 Da and a new cleavage site at Asp330. The observation of

ions at m/z 723.342+ and 482.563+ pertaining to this peptide in the N-deglycosylated

sample indicated that Ser332-334 were not O-glycosylated.

Figure 5. O-glycosylation of Mouse ZP3. (A). A peptide

(144)QGDVSSHPIQPTWVPFR(160) was present in the N/O-deglycosylated sample of

ZP3. The ions at m/z 976.02+ and 650.983+ corresponding to the above peptide without

any O-sugars were present with an Asn-Asp conversion at position 146. The CID

spectrum of m/z 976.02+ confirmed the sequence of this peptide; (B). Additional ions at

m/z 1158.562+ and 772.713+, which shifted the mass of this peptide up by 365.13 Da

(i.e., the mass of O-linked Galβ(1-3)GalNAc), were detected in the same sample. CID

spectrum of 772.713+ showed the presence of sequence ions as well as carbohydrates

marker ions. This observation implies that one of the three potential sites (Ser148,

Ser149, Thr155) was O-glycosylated. Upon endo-O-glycosidase treatment, which

removes the Galβ(1-3)GalNAc core, these two ions disappeared.

Figure 6. Summary of Mouse ZP1. The primary amino acid sequence (single letter

code) of ZP1 obtained from the native mouse zona pellucida extends from an N-

terminal pyroglutamine (pyrQ21) to a C-terminal arginine (R546) immediately upstream

of a dibasic cleavage site. There are 21 cysteine residues (yellow on blue background);

10 are in the ‘zona domain’ (yellow background) of which eight are conserved (C272,

C306, C325, C366, C449, C470, C522, C527). One disulfide bond was experimentally

determined, C449/C470 (solid line). All four of the potential N-linked sites (white on


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green background) were glycosylated (N49, N68, N240, N371). Peptides representing

~44% of mature ZP1 were not identified (white on grey backgrounds) because of

paucity of biological material. Within these sequences were multiple serine (S) or

threonine (T) residues representing potential O-linked glycosylation sites.



terminal valine (V35) to a C-terminal serine (S633) immediately upstream of a dibasic

cleavage site. There are 20 cysteine residues (yellow on blue background); 10 are in

the ‘zona domain’ (yellow background) of which eight are conserved (C365, C396,

C417, C457, C538, C608, C613, C623). Four disulfide bonds were experimentally

ascertained, C365/C457, C396/C417, C608/C613, C623/C627 (solid line). Among the

10 cysteine residues in the N-terminus of ZP2, the disulfide linkage of one (C84/C102)

was determined. Six of the six potential N-linked sites (white on green background) are

glycosylated (N83, N172, N184, N217, N264, N393). Peptides representing ~4% of

mature ZP2 were not identified (white on grey backgrounds). Within these sequences

was a single potential O-linked glycosylation site (T455).



terminal pyroglutamate (pyrQ23) to a C-terminal asparagine (N351) immediately

upstream of a dibasic cleavage site. There are eight conserved cysteine (yellow on blue

background) residues in the ‘zona domain’ (yellow background) that are disulfide linked,


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C46/C139, C78/C98, C216/C283, C240/C301 (solid line) as well four cysteines (C320,

C322, C323, C328) that are C-terminal to the ‘zona domain’. The linkage of the latter

(dotted lines) was indeterminate due to clustering of cysteine residues and the absence

of appropriate cleavage sites. Five of the six potential N-linked sites (white on green

background) are glycosylated (N146, N273, N304, N327, N330, but not N227) and there

appear to be two clusters of O-linked glycans at the N-terminus (predicted at T32, T34,

S39) and within the ‘zona domain’ (predicted at T155, T162). Clusters are indicated by

bracket, potential sites by asterisks and number of glycans by Arabic number.


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Table I. Disulfide Bond Linkage Mapping of Native Mouse Zona Proteins

ZP Residue# Sequence Enzyme m/z exp. m/z calc. 1 438-473^ TDPSLVLLLHQCWATPTT

SPFEQPQWPILSDGCPFK

Trypsin 1351.053+

1013.504+ 1351.003+

1013.504+

2 69-92

97-105

WNPSVVDTLGSEILN*CTYALDLER

FPYETCTIK

Trypsin 1269.953+

952.714+

762.355+

1269.943+

952.714+

762.375+ 2 83-88

97-105

N*CTYAL

FPYETCTIK

Asp-N +Trypsin

892.442+

595.293+ 892.402+

595.273+

2 362-367

457-462

DELCAQ

CYYIR

Asp-N +Trypsin

696.832+

464.883+ 696.802+

464.873+

2 382-401

410-420

PALNLDTLLVGN*SSCQPIFK

FHIPLNGCGTR

Trypsin 836.414+ 836.434+

2 599-632^ GLSSLIYFHCSALICNQVSLDSPL

CSVTCPASLR

Trypsin 1198.593+

899.194+ 1198.593+

899.204+

2 619-632^ DSPLCSV

TCPASLR

Asp-N +Trypsin

723.872+

723.842+

3 44-57

133-140

VECLEAELVVTVSR

VEVPIECR

Asp-N +Trypsin

830.123+ 622.834+

830.103+ 622.824+

3 65-81

91-101

LVQPGDLTLGSEGCQPR

FNAQLHECSSR

Trypsin 1020.163+

765.374+ 1020.153+

765.374+

3 70-81

91-101

DLTLGSEGCQPR

FNAQLHECSSR

Asp-N +Trypsin

855.393+ 641.784+

855.393+ 641.804+

3 214-224

277-286

DHCVATPSPLP

NTLYITCHLK

Asp-N +Trypsin

780.353+

585.524+ 780.393+

585.544+

3 306-342^ TSQSWLPVEGDADICDCC

SHGN*CSN*SSSSQFQIHGPR

Trypsin 988.414+ 988.424+

^ indicates intramolecular disulfide bonds within the same proteolytic fragment. N* represents an originally N-glycosylated asparagine residue converted to an aspartic acid upon PNGase F treatment.


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Table IIA. LC-MS Analysis of Mouse ZP1 N-linked Glycosylation Sites

Residue# Sequence Enzymes m/z exp. m/z calc.

39-55 GM*QLLVFPRPN*QTVQFK Trypsin 674.003+ 673.703+

49-55 N*QTVQFK Trypsin+Asp-N

433.232+ 432.732+

58-67 DEFGNRFEVN(N*CS) Asp-N 613.772+ 613.782+

228-244 C*QVASGHIPC*MVN*GSSK Trypsin 611.603+ 611.283+ 368-398 C*IFN*ASDFLPIQASIFSPQPPAPVTQSGPLR Trypsin 1119.533+ 1119.243+

C* = Carbamidomethylated cysteine M*= Methionine sulfoxide N*XS/T = N-linked asparagine converted to aspartate after PNGase F treatment (+0.984 Da) N = Non-N-linked asparagine site


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Table IIB. LC-MS Analysis of Mouse ZP2 N-linked Glycosylation Sites

Residue# Sequence Enzymes m/z exp. m/z calc.

69-92 WNPSVVDTLGSEILN*C*TYALDLER Trypsin 1383.652+ 922.733+

1383.182+ 922.453+

166-181 LADENQN*VSEM*GWIVK Trypsin 925.422+ 924.942+ 168-181 DENQN*VSEMGWIVK Trypsin 825.342+ 824.892+ 172-183 N*VSEM*GWIVKIG Asp-N 675.342+ 674.852+ 182-187 IGN*GTR Trypsin 309.652+ 309.172+ 184-194 (G)N*GTRAHILPLK (D) Asp-N 407.573+ 407.253+

217-246 N*ATGIVHYVQESSYLY

TVQLELLFSTTGQK Trypsin+Asp-N

1130.893+ 1130.583+

217-236 N*ATGIVHYVQESSYLYTVQL Trypsin+Asp-N

1143.562+ 1143.082+

264-282 N*ATHMTLTIPEFPGKLESV (D) Asp-N 696.013+ 695.693+ 264-278 N*ATHMTLTIPEFPGK Trypsin+

Asp-N 829.412+

553.273+ 828.922+

552.953+ 382-401 PALNLDTLLVGN*SSC*QPIFK Trypsin 1094.582+ 1094.082+ 393-401 N*SSC*QPIFK Trypsin+

Asp-N 541.252+ 540.762+

C* = Carbamidomethylated cysteine M*= Methionine sulfoxide N*XS/T = N-linked asparagine converted to aspartate after PNGase F treatment (+0.984 Da) N = Non-N-linked asparagine site


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Table IIC. LC-MS Analysis of Mouse ZP3 N-linked Glycosylation Sites

Residue# Sequence Enzymes m/z exp. m/z calc. 144-160 QGN*VSSHPIQPTWVPFR Trypsin 976.002+

650.983+ 975.502+ 650.673+

225-235 DPNSSPYHFIV Asp-N 638.292+ 638.302+ 214-235 DHCVATPSPLPDPNSSPYHFIV Asp-N 817.373+ 817.393+ 257-276 PRPETLQFTVDVFHFAN*SSR Trypsin 783.713+

588.034+ 783.403+

587.804+ 259-276 PETLQFTVDVFHFAN*SSR Trypsin 1048.522+

699.333+ 1048.022+

699.013+ 295-303 DKLNKAC*SF (N*KT) Asp-N 541.762+ 541.762+ 300-305 AC*SFN*K(T) Trypsin 727.301+

364.162+ 726.321+ 363.672+

306-342 TSQSWLPVEGDADIC*DC*C*SHG N*C*SN*SSSSQFQIHGPR

Trypsin 1046.434+

1046.164+(^) 1045.944+

330-342 N*SSSSQFQIHGPR Trypsin+Asp-N

723.342+ 482.563+

722.852+ 482.243+

C* = Carbamidomethylated cysteine N*XS/T = N-linked asparagine converted to aspartate after PNGase F treatment (+0.984 Da) N = Non-N-linked asparagine site ^ indicates the presence of an additional ion at m/z 1046.164+ that represents the charged N-deglycosylated species of ZP3 peptide (306)TSQSWLPVEGDADICDCC SHGNCSNSSSSQFQIHGPR(342) at either Asn327 or Asn330.


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Figure 1


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Figure 3


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Figure 4


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CHO (N240)

CHO (N49)

CHO (N68)

pyrQ21

R546

61

121

181

241

301

361

481

541

421

CHO (N371)

Mouse ZP1

QRLHLEPGFEYSYD GVRGMQLLVFPRP VQFK DEF

GNRFEVN G HVLEK VFIQAVLPNGR

SVGTHLPQER

SK

GPQGSITR

IF DFLPIQASIFSPQPPAPVTQSGPLR IATDKTFSSYYQGSDYPL

VRLLREPVYVEVRLLQRTDPSLVLLLHQ WATPTTSPFEQPQWPILSDG PFK DNYRTQ

VVAADKEALPFWSHYQRFTITTFMLLDSSSQNALRGQVYFF SASA HPLGS D

SGIARR

C

C C C

C

C C

C CC C C

C C

C

C C

C C C C

NQT VL

N SI YHWVTSEAQEHTVFSADYK DGRFHLR VDIA

QDVTLI PKPDHTVTPDPYLAPPTTPEPFTPHAFALHPIPDHTLAGSGHTGLTTLYPEQS

FIHPTPAPPSLGPGPAGSTVPHSQWGTLEPWELTELD

GS EA QQAG YDSTKEEP YYGNTVTLQ FK DNVHLA

YAPNG PPTQKTSAFVVFHVPLTL GTAIQVVGEQLIYENQLVSDI DS

AFRLHVR NAS LELR

G

DT STT

QVASGHIP MVN

SGYFTLVMSQETALTHGVLL

DVQK

Figure 6


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CHO (N393)

CHO (N184)

CHO (N264)

CHO (N217)

CHO (N172)

VSLPQSENPAFPGTLI DEVRIEF

SSRFDMEKWNPSVVDTLGSEIL YALDLERFVLKFPYET TIKVVGGYQVNIRVGDTT

TDVR EIVV R DLISFSFPQLFSRLADENQ EMGWIV

KIG RAHILPLKDAIVQGFNLLIDSQKVTLHVPA GIVHYVQESSYLYTVQLELLF

STTGQKIVFSSHAI APDLSVA HMTLTIPEFPGKLESVDFGQWSIPEDQWHANGID

KEATNGLR SLL PSEK PFYQFYLSSLKLTFYFQGNMLSTVIDPE H ESPVS

IDEL AQ DFEVYSHQTKPALNLDTLLVG QPIFKVQSVGLARFHIPLNG GTR

QKFEGDKVIYENEIHALWENPPSNIVFRNSEFR YYIRDSMLLNAHVKGHPSPEAF

VKPGPLVLVLQTYPDQSYQRPYR DEYPLVRYLRQPIYMEVKVLSRNDPNIKLVLDD WA

TSSEDPASAPQWQIVMDG EYELDNYRTTFHPAGSSAAHSGHYQRFDVKTFAFVSEARGL

SSLIYFH SALI NQVSLDSPL SVT PASLRS

C

C C

C C

C C

C C C

C C C

C

C

C

C C C C

DK

N T

YKDDMYHFF R NVS

NGT NAT

NAT

LNFRK

DGFM NSS

MTVR

PAIQAETHEIS

KTK

K

61

121

181

241

301

361

481

541

601

421

CHO (N83)

Mouse ZP2

V35

S633

*

Figure 7


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CHO (N304) CHO (N327) CHO (N330)

CHO (N273)

CHO (N146)

61

121

181

241

301

Mouse ZP3

pyrQ23

N351

QTLWLLPGGTPTPVGSSSPVKVE LEAELVVTVSRDLF

GTGKLVQPGDLTLGSEG QPRVSVDTDVVRFNAQLHE SSRVQMTKDALVYSTFLLHDPR

PVSGLSILRTNRVEVPIE RYPRQG SHPIQP WVPFRATVSSEEKLAFSLRLMEENW

NTEKSAPTFHLGEVAHLQAEVQTGSHLPLQLFVDH VATPSPLPDP PYHFIVDFHG

LVDGLSESFSAFQVPRPRPETLQFTVDVFHFA RNTLYIT HLKVAPANQIPDKLNKA

SF SQSWLPVEGDADI D SHG SSQFQIHGPRQWSKLVSRN

C

C C

C

C C

C

C C CC C

NVS

NSS

NSS

NKT N SNSS

T

3

2

*

* *

* ***

****

Figure 8


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Supplemental Table IA. LC-MS and MS/MS Analysis of Mouse ZP1.

Residueχ Sequences Enzymes m/z exp. m/z calc. Elution time (min)

21-33 qRLHLEPGFEYSY Asp-N 811.372+ 811.392+ 45.2

34-38 DC*GVR Trypsin+Asp-N 606.321+ 303.662+

606.281+ 303.642+

17.7

39-55 GM*QLLVFPRPN*QTVQFK Trypsin 674.003+ 673.703+

50.5

49-55¥ N*QTVQFK Trypsin+Asp-N 433.232+ 432.732+ 25.6

58-67 DEFGNRFEVN(N*CS) Asp-N 613.772+ 613.782+ 36.6

92-98 GC*HVLEK Trypsin 421.722+ 421.712+ 29.0 106-116 VFIQAVLPNGR Trypsin 607.342+ 607.362+ 40.4 218-227 SVGTHLPQER Trypsin 375.203+ 375.203+ 20.3 228-244¥ C*QVASGHIPC*MVN*GSSK Trypsin 611.603+ 611.283+ 29.7 275-294 SGYFTLVMSQETALTHGVLL Trypsin+Asp-N 1084.052+ 1084.062+ 61.3 347-358 DVQKGPQGSITR Asp-N 429.233+ 429.233+ 22.5 368-398¥ C*IFN*ASDFLPIQASIFSPQP

PAPVTQSGPLR Trypsin 1119.533+ 1119.243+ 62.7

403-407 IATDK Trypsin 547.311+ 547.311+ 20.9

408-422 TFSSYYQGSDYPLVR Trypsin 891.932+ 891.922+ 40.4 423-433 LLREPVYVEVR Trypsin 458.273+ 458.273+ 34.5 434-437 LLQR Trypsin 529.351+ 529.351+ 19.2 438-473^ TDPSLVLLLHQCWATPTT

SPFEQPQWPILSDGCPFK

Trypsin 1351.053+

1013.504+ 1351.003+

1013.504+ 58.2

475-484 DNYRTQVVAA Asp-N 568.792+ 568.792+ 27.5 479-497 TQVVAADKEALPFWSHYQR Trypsin 749.383+ 749.383+ 41.6 498-515 FTITTFMLLDSSSQNALR Trypsin 1023.012+ 1023.022+ 57.7 516-532 GQVYFFC*SASAC*HPLGS Trypsin+Asp-N 944.402+ 944.412+ 53.3 540-545 DSGIARR Asp-N 774.411+

387.722+ 774.421+

387.722+ 17.0

q = Pyroglutamic acid C* = Carbamidomethylated cysteine; C-C = disulfide bridge M*= Methionine sulfoxide N* = N-linked asparagine converted to aspartate after PNGase F treatment (+0.984 Da) χ is the residue number based on the primary sequence of full-length ZP1 ¥ represents N-linked peptides

^ represents disulfide bridged peptides


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Supplemental Table IB. LC-MS and MS/MS Analysis of Mouse ZP2.

Residuesχ Sequences Enzymes m/z exp. m/z calc. Elution time (min)

35-51 VSLPQSENPAFPGTLIC* Asp-N 915.462+

915.462+

48.1

54-64 DEVRIEFSSRF Asp-N 692.842+

462.223+ 692.852+

462.233+ 34.4

65-74 DMEKWNPSVV Asp-N 602.782+ 602.792+ 39.9 69-92¥ WNPSVVDTLGSEILN*C*TYALDLER Trypsin 1383.652+

922.733+ 1383.182+ 922.453+

67.6

93-96 FVLK Trypsin 506.341+ 506.331+ 26.0 97-105 FPYETC*TIK Trypsin 579.782+ 579.782+ 31.9

106-115 VVGGYQVNIR Trypsin 552.802+ 552.812+ 30.5 116-124 VGDTTTDVR Trypsin 482.242+ 482.242+ 19.7 134-151 C*PAIQAETHEISEIVVC*R Trypsin 1056.522+

704.683+ 1056.512+

704.683+ 65.6

153-165 DLISFSFPQLFSR Trypsin 778.912+ 778.912+ 62.3 166-181¥ LADENQN*VSEM*GWIVK Trypsin 925.432+ 924.942+ 37.4 182-187¥ IGN*GTR Trypsin 309.662+ 309.172+ 17.0 188-194 AHILPLK Trypsin 396.272+ 396.262+ 28.0 195-209 DAIVQGFNLLIDSQK Trypsin 830.952+ 830.952+ 53.9 206-216 DSQKVTLHVPA(N*) Asp-N 597.822+ 597.832+ 32.1 217-246¥ N*ATGIVHYVQESSYLY

TVQLELLFSTTGQK Trypsin+Asp-N 1130.893+ 1130.583+ 69.5

247-257 IVFSSHAIC*AP Trypsin+Asp-N 601.312+ 601.312+ 39.3 258-263 DLSVAC* Asp-N 664.291+ 664.301+ 26.8 264-282¥ N*ATHMTLTIPEFPGKLESV Asp-N 696.013+ 695.693+ 51.6 283-291 DFGQWSIPE Asp-N 539.742+ 539.752+ 47.7 279-302 LESVDFGQWSIPEDQWHANGIDK Trypsin 891.093+ 891.093+ 49.6 303-308 EATNνGLR Trypsin 381.202+ 380.702+ 18.4 314-317 SLLK Trypsin 460.321+ 460.311+ 20.8 318-323 TKPSEK Trypsin 689.391+

345.212+ 689.381+

345.202+ 20.4

324-335 C*PFYQFYLSSLK Trypsin 776.892+ 776.882+ 52.7 336-349 LTFYFQGNMLSTVI Trypsin+Asp-N 817.412+ 817.422+ 58.5 350-361 DPEC*HC*ESPVSI Trypsin+Asp-N 715.302+ 715.292+ 33.4 362-367 DELC*AQ Asp-N 735.301+

368.152+ 735.301+

368.152+ 21.2

372-386 DFEVYSHQTKPALNL Asp-N 587.953+

881.432+ 587.973+

881.442+ 42.1

382-401¥ PALNLDTLLVGN*SSC*QPIFK Trypsin 1094.582+

730.033+ 1094.082+

729.723+ 56.5

402-409 VQSVGLAR Trypsin 415.252+ 415.252+ 22.8 410-420 FHIPLNGC*GTR Trypsin 636.322+ 636.322+ 33.1 421-427 FEGDK Trypsin 595.281+ 595.271+ 18.8 428-448 VIYENEIHALWENPPSNIVFR Trypsin 847.443+ 847.443+ 54.3 449-453 NSEFR Trypsin 326.662+ 326.662+ 18.8


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458-462 C*YYIR Trypsin 387.682+ 387.682+ 26.6 463-472 DSM*LLNAHVK Trypsin 572.292+ 572.302+ 25.7 473-500 GHPSPEAFVKPGPLVLVLQTYPDQ

SYQR

Trypsin 1041.893+

781.654+ 1041.883+

781.664+ 52.0

473-503 GHPSPEAFVKPGPLVLVLQTYPDQSYQRPYR

Trypsin 1180.613+

885.714+ 1180.623+

885.724+ 52.4

504-511 KDEYPLVR Trypsin 510.282+ 510.282+ 24.5 512-514 YLR Trypsin 451.261+ 451.271+ 17.7 515-522 QPIYM*EVK Trypsin 512.262+ 512.262+ 25.2 523-526 VLSR Trypsin 407.302+ 407.302+ 16.7 527-532 NDPNIK Trypsin 350.682+ 350.692+ 17.8 533-567 LVLDDC*WATSSEDPASAPQWQIV

MDGC*EYELDNYR Trypsin 1378.583+

1034.184+ 1378.603+

1034.204+ 55.4

545-556 DPASAPQWQIVM Asp-N 671.822+ 671.832+ 49.1 568-585 TTFHPAGSSAAHSGHYQR Trypsin 637.963+

478.724+ 637.973+

478.734+ 20.1

586-589 FDVK Trypsin 508.281+ 508.281+ 21.0 590-598 TFAFVSEAR Trypsin 514.272+ 514.262+ 35.7 599-632 GLSSLIYFHCSALICNQVSLDSPL

CSVTCPASLR Trypsin 1198.593+

899.194+ 1198.593+

899.204+ 58.5

619-633 DSPLC*SVTC*PASLRS Asp-N 825.382+ 825.392+ 39.0 C* = Carbamidomethylated cysteine; C-C = disulfide bridge M*= Methionine sulfoxide N* = N-linked asparagine converted to aspartate after PNGase F treatment (+0.985 Da) χ is the residue number based on the amino acid sequence of full-length ZP2 ¥ represents N-linked peptides ν represents deamidated asparagine residues


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Supplemental Table IC. LC-MS and MS/MS Analysis of Mouse ZP3.

Residuesχ Sequences Enzymes m/z exp. m/z calc. Elution time (min)

23-43 qTLWLLPGGTPTPVGSSSPVK Trypsin 702.423+ 527.064+

702.383+ 527.044+

55.7

23-43ξ qTLWLLPGGT*PT*PVGSS*SPVK Trypsin 1067.473+

1067.513+

45.7

44-57 VEC*LEAELVVTVSR Trypsin 802.422+

535.273+ 802.422+

535.283+ 47.3

58-64 DLFGTGK Trypsin 369.192+ 369.202+ 29.4 65-81 LVQPGDLTLGSEGC*QPR Trypsin 913.942+ 913.962+ 36.1 82-90 VSVDTDVVR Trypsin 989.541+

495.282+ 989.521+ 495.272+

26.6

91-101 FNAQLHEC*SSR Trypsin 674.802+ 450.203+

674.812+ 450.213+

22.5

102-106 VQMTK Trypsin 303.662+ 303.662+ 17.8 107-129 DALVYSTFLLHDPRPVSGLSILR Trypsin 857.143+

643.104+ 857.143+

643.114+ 55.3

130-140 TNRVEVPIEC*R Trypsin 458.233+ 458.243+ 26.8 133-140 VEVPIEC*R Trypsin 501.252+ 501.262+ 27.6 141-143 YPR Trypsin 435.231+ 435.231+ 17.4 144-160¥ QGN*VSSHPIQPTWVPFR Trypsin 976.002+

650.983+ 975.50+2 650.67+3

39.9

144-160¥ξ QGN*VSSHPIQPT*WVPFR Trypsin 1158.572+

772.703+ 1158.072+ 772.38+3

39.9

161-168ξ AT*VSSEEK Trypsin 608.272+ 608.282+ 16.7 169-174 LAFSLR Trypsin 353.722+ 353.722+ 36.5 175-184 LMEENWNTEK Trypsin 647.302+ 647.302+ 29.4 185-192 SAPTFHLG Trypsin+Asp-N 415.212+ 415.212+ 25.0 193-213 EVAHLQAEVQTGSHLPLQLFV Asp-N 772.743+ 772.753+ 53.3 214-224 DHC*VATPSPLP Asp-N 597.282+ 597.282+ 33.7 214-235 DHC*VATPSPLPDPNSSPYHFIV Asp-N 817.373+ 817.393+ 48.6 225-235 DPNSSPYHFIV Asp-N 638.292+ 638.302+ 44.5 236-242 DFHGC*LV Asp-N 424.192+ 424.192+ 35.2 243-256 DGLSESFSAFQVPR Trypsin+Asp-N 770.37+2 770.38+2 47.3 257-276¥ PRPETLQFTVDVFHFAN*SSR Trypsin 783.71+3

588.03+4 783.40+3

587.80+4 53.1

277-286 NTLYITC*HLK Trypsin 631.83+2 631.83+2 32.0 287-297 VAPANQIPDK Trypsin 526.78+2 526.79+2 22.0 295-303 DKLNKAC*SF(N*) Asp-N 541.76+2 541.76+2 26.1 300-305¥ AC*SFN*K Trypsin 727.30+1

364.16+2 726.32+1 363.67+2

19.3

306-342¥ TSQSWLPVEGDADIC*DC*C*SHGN*C*SN*SSSSQFQIHGPR

Tryspin 1046.434+

1046.164+&

1045.944+ 40.2

330-342¥ N*SSSSQFQIHGPR Tryspin+Asp-N 723.34+2 722.85+2 30.2


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482.56+3 482.24+3 330-351¥ N*SSSSQFQIHGPRQWSKLVSRN Asp-N 848.75+3

636.80+4 848.43+3 636.58+4

40.3

q = Pyroglutamic acid C* = Carbamidomethylated cysteine N* = N-linked asparagine converted to aspartate after PNGase F treatment (+0.985 Da) T* & S* represent O-glycosylated serine and threonine residues χ represents the residue number based on the primary sequence of full-length ZP3 ξ represents O-linked peptides ¥ represents N-linked peptides & indicates the presence of a singly N-deglycosylated species either at Asn327 or Asn330


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Emily S. Boja, Tanya Hoodbhoy, Henry M. Fales and Jurrien Deanspectrometry

Structural characterization of native mouseZona pellucida proteins using mass

published online June 10, 2003J. Biol. Chem.

10.1074/jbc.M304026200Access the most updated version of this article at doi:

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corresponding author: dr. emily s. boja laboratory of ...structural characterization of native mouse...

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