Reconstitution of Signaling Events of Egg Fertilization

In vitro reconstitution system involves both rafts and CSF extracts prepared from Xenopus eggs, and a series of biochemical and cell biological events associated with fertilization can be examined (Fig. 1).

See section 3.1

CSF _ extracts'

TX-100 extraction of eggs

Sue rose-density gradient fractionation

Recovery of LD-DIM (rafts)

Crush spin of unfertilized eggs Recovery of cytoplasm (CSF extracts)

Freeze in liquid nitrogen One-time thawing

See subheading 3.2

Reconstitution of fertilization signaling

Variations of strategies

Sperm-egg membrane interaction

Activation of Src

Addition of activators/inhibitors

Phosphorylation of PLCy

Use of rafts from activated eggs

IP3 production

Immunodepletion

Calcium release

Use of rafts from culture cells

De phosphorylation of MAP kinase

Cell cycle transition

Transiational control of maternal mRNAs

Zygotic reprogramming

Output

Identification of novel components of fertilization (e.g. membrane receptor for sperm; a novel Src substrate See subheading 3,3)

Better understanding of fertilization system as a whole (e.g. global protein analysis in the course of fertilization)

Fig. 1. Schematic representation of a reconstitution system of Xenopus egg fertilization signaling. This system involves the membrane/lipid rafts and cell-free extract that are prepared from unfertilized eggs. By combining these two different egg fractions, a series of events associated with fertilization, from the sperm-egg membrane interaction through the Ca2+-depen-dent cell cycle transition, can be reproduced in vitro. Because of its cell-free nature, this system enables us to perform specific targeting of signaling molecules of interest (e.g., pharmacological activation or activation, immunochemical depletion) that would contribute to identifying crucial components for fertilization signaling. A systematic as well as a pin-point approach to characterize egg proteins with use of mass spectrometric analysis certainly would be helpful to validate this strategy.

3.2.1. In Vitro Protein-Tyrosine Kinase Assay of the Isolated Raft Fraction

1. Raft fractions (10 ||L, an equivalent of 30-60 eggs) were preincubated with or without activators such as 106/mL sperm, 1 mM GTPyS, 1 mM cAMP, 1 mM RGDS peptide, or 1 mM CaCl2 at a final volume of 12.5-25 |L for 10 min at 30°C.

2. The mixtures were further incubated at 30°C for 10 min in the presence of 5 mM MgCl2, 20 mM Tris-HCl, 1 mM DTT, 2 |M [y-32P]ATP (10 |Ci), and 1 mM Cdc2 peptide at a final volume of 50 | L.

3. The reaction was stopped by the addition of 50 |L of Laemmli's SDS sample buffer followed by heat treatment at 98°C for 5 min.

4. Phosphorylated samples were separated by SDS polyacrylamide gel electrophoresis (PAGE) on 16% gels (see Subheading 3.3.3.). 32P-labeled Cdc2 peptide was visualized and quantified by a BAS2000 Bioimaging analyzer (Fuji Film).

3.2.2. Tyrosine Phosphorylation of Src, Phospholipase Cg, and MAPK

(see Note3)

1. Rafts alone (3 |L: 10-20 egg equivalent), CSF extracts alone (27 |L: 30 egg equivalent), or a mixture of the both are incubated in the absence or presence of 1.5 |L of the activators (e.g., sperm) or 1.5 |L of various inhibitors for 10 min at 30°C. Inhibitors used are 10 |M PP2, PP3, U73122, or U73343; 100 |M heparin; or 5 mM EGTA.

2. The mixtures are then incubated with 5 mM MgCl2 and 1 mM ATP for 10 min at 30°C.

3. The kinase reaction was terminated by the addition of 30 |L of 10 mM EDTA on ice.

4. Proteins are solubilized by incubation in the presence of 0.1% SDS and 1 mM sodium orthovanadate at 37°C for 10 min and collected as the supernatant fractions after centrifu-gation at 150,000g for 10 min at 4°C.

5. To assess tyrosine phosphorylation of Src and MAPK, protein samples (10-30 |g) are separated by SDS-PAGE on 8% gels and analyzed by immunoblotting (see below) with either antiphospho Src Y416 antibody (1 |g/mL) or antiphospho MAPK antibody (0.5 |g/mL). For tyrosine phosphorylation of PLCy, protein samples (50-500 |g) are subjected to immunoprecipitation with anti-PLCy antibody (1 |g/mL) followed by immunoblotting with antiphosphotyrosine antibody PY99 (1 |g/mL) or anti-PLCy antibody (0.5 |g/mL).

3.2.3. Immunoprecipitation, SDS-PAGE, and Immunoblotting

1. Protein samples (50-500 | g at 1 | g/| L) are immunoprecipitated with 1-5 | L appropriate antibody for 3 h at 4°C.

2. After centrifugation at 10,000g for 10 min at 4°C, the immune complexes are adsorbed onto 10 |L protein A-Sepharose beads by gentle agitation for 30 min at 4°C.

3. The beads are washed three times with 500 | L RIPA buffer, washed once with extraction buffer containing 1% Triton X-100, and used as immunoprecipitates.

4. The immunoprecipitates prepared as above are treated with Laemmli's SDS sample buffer at 98°C for 5 min.

5. SDS-PAGE and immunoblotting of the SDS-denatured proteins are done as described previously using SDS-PAGE apparatus (Atto, Tokyo, Japan) and semidry blotting apparatus (Bio-Rad; Hercules, CA), respectively.

3.2.4. Calcium Release Assay

1. Ultraviolet-excitable ratiometric fluorescent calcium indicator Fura-2 is added to the CSF extracts at the time 3 to 10 min prior to the initiation of reconstitution of signaling events to a final concentration of 2 | M.

2. The level of free calcium in the extracts is continuously monitored by ratio-imaging microscopy using the high-frame digital CCD imaging ARGUS/HISCA system from Hamamatsu Photonics. Excitation wavelengths are set at 340 and 380 nm; monitoring of the emission was made at 510 nm. The basal level of fluorescent signal was monitored over several minutes at 21 to 23°C prior to the initiation of the reactions.

3. The reaction was initiated by the administration of the effectors (e.g., rafts) that had been preincubated in the absence or the presence of various inhibitors or activators.

4. Further monitoring of the fluorescent signal was made at intervals of 10 to 15 s. Initial time of the calcium rise, its peak height, and duration of the rise are determined. Samples that showed no calcium rise during 40 min of the reaction are recorded as negative. Calcium rise is defined for the fluorescent signal that gives more than 0.04 unit of the 340/380 ratio, which persists for more than 30 s. Table 1 summarizes results of calcium release assay obtained with several conditions involving rafts, activators/inhibitors, and CSF extracts.

3.2.5. Cell Cycle Transition Assay

1. Demembranated sperm nuclei (1 ||L: a final concentration of 105/mL) are added to the CSF extracts (27 |L) on ice prior to the initiation of reconstitution experiments.

2. Reaction is initiated by the addition of raft fractions as described above. Nuclear morphology of the added sperm nuclei are observed and scored by fluorescent microscopy after withdrawing 1 |L of extract at appropriate time intervals and adding 4 |L 0.33X modified Ringer's solution containing 1 |g/mL Hoechst 33342 (Sigma), 10% formaldehyde, and 50% glycerol.

3.2.6. Immunodepletion of Phospholipase Cg From CSF Extracts

1. Depletion of PLCy is done by incubating CSF extracts (100 |L) with anti-PLCy antibody (1 |L) for 3 h at 4°C followed by the adsorption of the immune complexes onto protein A-Sepharose beads.

2. The resulting supernatant fractions are used as CSF extracts depleted of PLCy (CSF/APLCy). The efficiency of the depletion is determined by densitometry scanning of the anti-PLCy immunoblotting results (see Subheading 3.3.3.) for intact CSF extracts and CSF/APLCy.

3.2.7. Effect of Cultured Cell Raft Fractions Containing Overexpressed Xenopus Src (see Note 4)

1. COS7 cells are maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum at 37°C in a humidified 5% CO2 atmosphere. Cells of 2030% confluence in 100-mm dishes are transfected with the expression vector of FLAG-epitope tagged Xenopus Src (2 |g DNA/dish) using Effectene™ reagent (Qiagen) according to the manufacturer's standard protocol.

2. The transfection treatment proceeds for 24 h at 37°C. The resultant transfected cells are harvested, extracted with raft buffer, and subjected to sucrose density gradient ultracen-trifugation as described above.

3. After ultracentrifugation, raft fractions are collected. The expression of Xenopus Src is verified by immunoblotting of the fractions with either anti-pepY antibody or anti-FLAG antibody.

4. The effect of the raft fraction containing Xenopus Src on the reconstitution of fertilization signaling events (e.g., tyrosine phosphorylation of PLCy, calcium release) is analyzed as described above.

Table 1

Effect of Rafts Incubated With Sperm or GTP7S on the Ca2+ Release in CSF Extracts

Table 1

Effect of Rafts Incubated With Sperm or GTP7S on the Ca2+ Release in CSF Extracts

%

Time

Peak

Duration

Activators/inhibitors/rafts

Ca2+ rise11

to Ca2+ rise6

amplitude6

of Ca2+ rise6

IP3 (10 ||M)

100 (8)

0.9 ± 0.1

0.44

± 0.04

2.4

± 0.2

+ heparin (500 ||M)

14 (7)

1.1 (1)

0.07

(1)

1.8

(1)

U rafts

20 (5)

2.1 (2)

0.04

(2)

2.8

(2)

U rafts + sperm (106/mL)

82 (11)

1.7 ± 0.2 (9)

0.08

± 0.11 (9)

5.5

± 1.1 (9)

+ PP2 (10 ||M)

13 (8)

2.1 (1)

0.04

(1)

2.6

(1)

+ PP3 (10 ||M)

100 (3)

2.0 ± 0.4 (3)

0.11

± 0.03 (3)

9.1

± 3.8 (3)

+ U-73122 (10 ||M)

50 (4)

17.2 (2)

0.06

(2)

1.8

(2)

+ U-73343 (10 ||M)

100 (2)

1.6

0.07

10.3

+ heparin (500 ||M)

0 (3)

-

-

-

U rafts + GTPyS (1 mM)

100 (3)

1.4 ± 0.4

0.04

± 0.01

3.4

± 0.1

U rafts + RGDS (1 mM)

0 (2)

-

-

-

F rafts

50 (4)

2.8 (2)

0.07

(2)

3.7

(2)

A rafts

0 (4)

-

-

-

H rafts

100 (6)

2.4 ± 0.2

0.20

± 0.03

6.5

± 1.1

+ PP2 (10 |M)

0 (5)

-

-

-

+ PP3 (10 |M)

100 (2)

2.1

0.30

13.0

+ peptide A7 (15 |M)

0 (3)

-

-

-

+ peptide A9 (15 |M)

67 (3)

2.3 (2)

0.19

(2)

23.3

(2)

+ U-73122 (10 |M)

20 (5)

2.9 (1)

0.07

2.8

+ U-73343 (10 |M)

100 (2)

3.2

0.28

10.2

CSF/APLCy + IP3 (10 |M)

100 (3)

0.6 ± 0.1

0.38

± 0.02

2.5

± 0.4

CSF/APLCy + U rafts

25 (4)

2.2 (1)

0.06

(1)

5.3

(1)

+ sperm (106/mL)

CSF/APLCy + H rafts

75 (4)

2.1 ± 0.6 (3)

0.24

± 0.03 (3)

5.2

± 0.9 (3)

+ PP2 (10 |M)

0 (3)

+ U-73122 (10 |M)

0 (4)

U rafts, rafts from unfertilized eggs; F rafts, A rafts, and H rafts indicate rafts from unfertilized, fertilized (5-min insemination), Ca2+ ionophore-activated (5-min treatment with A23187), and H2O2-activated eggs (5-min treatment), respectively. CSF/APLCy indicates CSF extracts depleted of PLCy. IP3, inositol 1,4,5-triphosphate.

aNumber of samples tested indicated in parentheses.

6Data (minutes in Time to Ca2+ rise and Duration of Ca2+ rise columns or ratio unit in Peak amplitude column) shown are means ± standard deviations of more than three independent experiments, average values of two experiments, or an exact value from one experiment, all of which are dependent on the numbers of samples with Ca2+ rise.

U rafts, rafts from unfertilized eggs; F rafts, A rafts, and H rafts indicate rafts from unfertilized, fertilized (5-min insemination), Ca2+ ionophore-activated (5-min treatment with A23187), and H2O2-activated eggs (5-min treatment), respectively. CSF/APLCy indicates CSF extracts depleted of PLCy. IP3, inositol 1,4,5-triphosphate.

aNumber of samples tested indicated in parentheses.

6Data (minutes in Time to Ca2+ rise and Duration of Ca2+ rise columns or ratio unit in Peak amplitude column) shown are means ± standard deviations of more than three independent experiments, average values of two experiments, or an exact value from one experiment, all of which are dependent on the numbers of samples with Ca2+ rise.

3.3. Identification of Raft-Associated Proteins by MS

To explore the role of protein-tyrosine phosphorylation in fertilization, we have been interested in identifying tyrosine-phosphorylated proteins in fertilized or parthe-nogenetically activated Xenopus eggs. We have demonstrated that Src, PLCy, and the adaptor protein Shc become tyrosine-phosphorylated following egg activation. We have found that a 30-kDa raft-associated protein is a major tyrosine-phosphorylated protein

Protein detection with SDS-PAGE/immunoblotting Staining and destaining for in-gel digestion In-gel reduction and alkylation of thiol group of Cys In-gel digestion with Achromobacter Protease I

Peptide extraction and mass spectrometric analysis *

Searching sequence databases using MS data Peptide mass fingerprinting Product ion mass fingerprinting EST database

UniGene and EST consensus sequence database

Fig. 2. Molecular identification of egg raft-associated proteins that are tyrosine-phosphory-lated at fertilization of Xenopus eggs by mass spectrometry and data mining. Immunoblotting analysis (IB) with antiphosphotyrosine antibody (anti-pTyr) of the raft fractions that was prepared from unfertilized eggs (Uf) and fertilized eggs (F, 5 min after insemination) revealed that in addition to Xenopus Src (xSrc), a 30-kDa protein is predominantly phosphorylated on tyrosine residue(s). According to the experimental scheme shown in this figure and described in the text, we have characterized this protein, termed pp30, and it turns out to be uroplakin III, a single-transmembrane protein that contains a tyrosine phosphorylation site in the carboxyl-terminal cytoplasmic region (20) and that is a potential target of sperm-derived protease, which is believed to be essential for sperm-induced egg activation (21).

in the membrane/lipid raft fraction of activated Xenopus eggs (Fig. 2). In this subheading, we describe molecular identification of such raft-associated proteins using MS, with special emphasis on how to use sequence database to identify unknown Xenopus gene product.

3.3.1. In-Gel Digestion for MS Identification of Proteins 3.3.1.1. Staining and Destaining for In-Gel Digestion

1. Visualize proteins in SDS polyacrylamide gels by the silver staining method using a Wako Silver Stain MS Kit for MS protein identification.

2. Excise bands or spots corresponding to proteins.

3. Destain proteins in gel pieces by treating with 0.1 mL of the destaining solution provided by the kit for 15 min according to the instructions.

4. Pull off solution and discard.

5. Add 0.2 mL 20 mM EDTA/100 mM NH4HCO3 and incubate at room temperature for 15 min.

6. Wash gel pieces by incubation with 1 mL water at room temperature for 3 min.

7. Pull off solution and discard.

8. Repeat steps 6 and 7.

3.3.1.2. In-Gel Reduction and Alkylation of Thiol Groups of Cys

1. Dry gel pieces in a centrifugal concentrator.

2. Prepare a fresh solution for reduction: 10 mM DTT/10 mM EDTA/100 mM NH4HCO3.

3. Cover dried gel pieces with the solution for reduction to allow dried gel pieces to swell at room temperature.

4. Reduce proteins in gel pieces at 50°C on a heating block for 1 h.

5. Remove tubes from a heating block and let cool to room temperature.

6. Pull off solution and discard.

7. Dry gel pieces in a centrifugal concentrator.

8. Prepare a fresh solution for alkylation: 40 mM iodoactamide/10 mM EDTA/100 mM NH4HCO3 and keep it in the dark.

9. Cover dried gel pieces with the solution for reduction to allow dried gel pieces to swell at room temperature.

10. Allow reaction to proceed in the dark at room temperature for 30 min.

11. Pull off solution and discard.

12. Wash gel pieces by incubation with 1 mL water at room temperature for 3 min.

13. Pull off solution and discard.

14. Repeat steps 12 and 13.

3.3.1.3. In-Gel Digestion With Achromobacter Protease I

1. Dry gel pieces in a centrifugal concentrator.

2. Prepare a stock solution of Achromobacter protease I (Lysyl endopeptidase®) with 0.05 ami-dase unit (AU) (~20 |lg) of enzyme/|L of 100 mM Tris-HCl, pH 8.9, and keep at 4°C.

3. Dilute 1 |L stock solution to 3 mL 100 mM Tris-HCl, pH 8.9, to prepare a working solution of Achromobacter protease I and keep on ice.

4. Swell dried gel pieces with working solution of Achromobacter protease I on ice.

3.3.1.4. Peptide Extraction and MS Analysis

1. Add 40 |L (or enough to cover gel pieces) 5% formic acid and incubate at room temperature for 10 min.

2. Centrifuge gel pieces, collect supernatant, and save it in on ice.

3. Add 40 |L (or enough to cover gel pieces) 50% acetonitrile/5% formic acid and incubate at room temperature for 10 min.

4. Centrifuge gel pieces, collect supernatant, and combine it with the supernatant from step 2.

5. Add 40 |L (or enough to cover gel pieces) of 95% acetnitrile/5% formic acid and incubate at room temperature for 10 min.

6. Centrifuge gel pieces, collect supernatant, and transfer it into a tube to which supernatant from step 4 has been added to supernatant from step 2.

7. Evaporate organic solvent and reduce volume by a centrifugal concentrator until desired volume for desalting with ZipTip (Millipore) or liquid chromatography/MS has been reached.

8. Analyze peptide fragments by MS followed by desalting with ZipTip or liquid chromatography.

3.3.2. Searching Sequence Databases Using MS Data

Searching amino acid or nucleotide sequence database by using MS data of in-gel digests of unknown proteins is well established as a method for the rapid identification of proteins. At present, several computer programs with different algorithms for protein identification by database searching using MS data have been developed. There are two major approaches for MS-based protein identification. One is the approach called peptide mass fingerprinting, and another is the product ion mass fingerprinting approach, which uses uninterpreted tandem mass spectrometry (MS/MS) data (12,13).

3.3.2.1. Peptide Mass Fingerprinting

In peptide mass fingerprinting, experimental data used for searching are observed mass values of peptide fragments obtained by the digestion of a protein by an enzyme with high residue specificity. Several search engines for peptide mass fingerprinting can be freely accessed across the World Wide Web at uniform resource locators (URLs). Some examples follow. It is possible to obtain licenses to operate some of them for your in-house server.

Peptide Mass Fingerprint in MASCOT by Matrix Science: http://www.matrixscience.com/ search_form_select.html

MS-fit in ProteinProspector by University of California, San Francisco: http://prospector. ucsf.edu/ucsfhtml4.0/msfit.htm

ProFound in PROWL by Rockefeller University: http://prowl.rockefeller.edu/profound_bin/ WebProFound.exe

ProFound in PROWL by Genomic Solutions: http://65.219.84.5/service/prowl/profound. html

PeptideSearch by European Molecular Biology Laboratory: www.narrador.embl-heidelberg.de/GroupPages/PageLink/peptidesearchpage.html

PeptIdent in ExPASy by Swiss Institute of Bioinformatics: http://us.expasy.org/tools/ peptident.html

PepMAPPER by the University of Manchester Institute of Science and Technology: http://wolf. bms.umist.ac.uk/mapper/

3.3.2.2. Product Ion Mass Fingerprinting

Another major approach for MS-based protein identification uses uninterpreted MS/MS data (product ion mass lists) from one or more peptide fragments obtained from the digestion of a protein by an enzyme with high residue specificity (14). In principle, this is similar to peptide mass fingerprinting. The experimental data are compared with calculated mass values of peptide fragments or product ions, obtained by applying appropriate cleavage rule of protease or gas phase fragmentation. Several tools for product ion mass fingerprinting are available on the Internet sites. Some examples follow. It is possible to run a local copy of some of them for your in-house server.

MS/MS Ions Search in MASCOT by Matrix Science: http://www.matrixscience.com/ search_form_select.html

MS-tag in ProteinProspector by University of California, San Francisco: http://prospector. ucsf.edu/ucsfhtml4.0/mstag.htm

PepFrag in PROWL by Rockefeller University: http://prowl.rockefeller.edu/PROWL/ pepfragch.html

Cocoozo (National Institute of Advanced Industrial Science and Technology): http:// www.cbrc.jp/cocoozo/

Sonor ms/ms™ in PROWL by Genomic Solutions: http://65.219.84.5/service/prowl/ sonar.html

3.3.2.3. Expressed Sequence Tag Database

The protein sequence database searched on servers for MS-based protein identification is mainly nr of the National Center for Biotechnology Information (NCBI) and SwissProt.

Unlike yeast and fly, all information on amino acid sequences of gene products of Xenopus is not registered in any database. Unfortunately, at present, the number of entries of Xenopus protein sequence in public databases is poor. The current MS-based protein identification approach largely depends on quality of sequence database. If a protein sequence database used in a search does not contain any information on sequence of an "unknown protein," a particular protein match will be missed.

As product ion mass fingerprinting using uninterpreted MS/MS data can be performed on short stretches of sequence, it is possible to search, instead of an amino acid sequence, a short nucleotide sequence from an expressed sequence tag (EST) that contains a "single-pass" cDNA sequence (15,16). In general, as the reading frame for translation is unknown, the nucleotide sequence is translated to an amino acid sequence in all six reading frames by a search engine prior to searching. If the MS/MS data did not match any sequence in protein databases, the same data set should be searched against an EST database.

A public EST database, dbEST, is a division of GenBank. Currently, NCBI has compiled three databases of est_human, est_mouse, and est_others from the dbEST. These three EST databases are included in available sequence databases on MASCOT (MS/MS ions search) and ProteinProspector (MS-tag) servers that can be freely accessed on Web sites. In general, the est_others database should be used for Xenopus protein identification. If you can access to an in-house server for product ion mass fingerprinting, your MS/MS data is able to search against independent EST databases of Xenopus obtained from various Xenopus EST projects.

3.3.2.4. UniGene and EST Consensus Sequence Database

EST databases are invaluable resources for protein identification and characterization. This allows novel proteins to be identified in the absence of entry in a protein sequence database. However, there are a few drawbacks of using an EST database in proteomics:

1. In general, there are very few long sequences, and each EST translation does not correspond to full-length protein. Therefore, if MS/MS data from multiple peptides derived from the same protein are searched against an EST database, peptide matches are scattered over a number of database entries. With limited exceptions, peptide mass fingerprinting cannot be applied to short stretches of EST.

2. An EST identification is not a real protein identification. Sequence information obtained from the EST is helpful for protein mining. However, it is hard to identify a novel protein using only the information obtained from the EST without using molecular biological strategy, which is time consuming and complicated.

There have been a number of attempts to identify unique genes and transcripts represented by EST data, such as UniGene of NCBI and The Institute of Genomic Research (TIGR) Gene Index (TGI). In both UniGene and TGI, EST sequences are grouped into a set based on overlapping sequence, in which each set theoretically represents a unique expressed gene. Such nonredundant sets are represented by sequence clusters in the UniGene and by assembled sequences (tentative consensus, TC) sequences in the TGI.

UniGene cluster is an index created by automatically partitioning GenBank sequences into a nonredundant set of gene-oriented clusters. Each UniGene cluster is a list of the nucleotide sequences registered in GenBank that represents a unique gene.

As there are very few long sequences in an EST database, in searching against the EST database using MS/MS data from multiple peptides derived from the same protein, peptide matches are scattered over a number of EST entries. It would be time consuming and not easy to find a connection among a number of matched EST entries. This difficulty can be improved with UniGene. There is an option to group-matched EST entries according to UniGene in one of the product ion mass fingerprinting search engines, a MS/MS ions search of MASCOT (17).

The other solution is using an EST consensus sequence database, instead of an EST database, as a database for product ion mass fingerprinting. The TIGR African clawed frog (Xenopus) Gene Index (XGI) uses assembly algorithms to produce consensus sequences (TC sequences) that represent the underlying mRNA transcripts (http:// www.tigr.org/tigr-scripts/tgi/T_index.cgi?species=xenopus). UniGene does not attempt to assemble EST sequences within each cluster to produce a consensus sequence. Xenopus TC sequence database is a set of virtual transcripts of Xenopus. Therefore, peptide mass fingerprinting can be applied to EST consensus sequences. The TC sequence database from XGI is an invaluable resource for protein identification and characterization in MS-based identification of Xenopus proteins.

4. Notes

1. This procedure takes about 30 s between the start of the wash treatment and freezing. Only batches of eggs that showed a successful egg activation rate of more than 80% within 20 min of the activation treatment were used for experiments. The successful egg activation rate was determined by monitoring the occurrence of cortical contraction of the pigmented area—a hallmark of successful egg activation—in another group of the same batch of eggs.

2. In the initial procedure, which we used for the preparation of egg raft fractions (10), egg samples were directly mixed with Triton X-100-containing buffer, homogenized, and fractionated. However, we have found that, under these conditions, the resulting raft fractions contained mitochondria or mitochondrial membranes, as judged by the detection of some mitochondria-associated proteins (e.g., cytochrome c oxidase subunits) with the use of MS. The present protocol, which utilizes detergent-free extraction of egg samples and isolation of low-density membrane fraction followed by its solubilization with Triton X-100, essentially excludes the contamination of such nonplasma membranous components in the raft fractions.

3. Tyrosine phosphorylation of Src, PLCy and MAPK could also be determined in rafts and nonrafts that had been prepared from unfertilized, fertilized, and parthenogenetically activated egg samples. In this case, 10 |lg of protein from the SDS-solubilized rafts and 500 |lg of proteins from the Triton X-100-soluble nonrafts were subjected to immunopre-cipitation and immunoblotting with the same antibodies used for in vitro reconstitution experiments (see refs. 18,19).

4. We also used the raft fractions of COS7 cells expressing different versions of Xenopus Src; wild type, kinase inactive, and kinase active. The kinase-inactive version was made by amino acid substitution of Lys-294 in the ATP-binding site with Met. The kinase-active version was made by substitution of Tyr-526 in the carboxyl-terminal tail to Phe. As expected, the COS7 cell rafts containing kinase-active Src induced calcium release in the CSF extracts, whereas those containing kinase-inactive Src did not show such effect (see ref. 19).

References

1 Masui, Y. (2000) The elusive cytostatic factor in the animal egg. Nat. Rev. Mol. Cell. Biol. 1, 228-232.

2 Iwao, Y. (2000) Mechanisms of egg activation and polyspermy block in amphibians and comparative aspects with fertilization in other vertebrates. Zool. Sci. 17, 699-709.

3 Yanagimachi, R. (1994) Fertilization, in The Physiology of Reproduction (Knobil, E. and Neill, J. D., eds.), Raven, New York, pp. 189-317.

4 Runft, L. L., Jaffe, L. A., and Mehlmann, L. M. (2002) Egg activation at fertilization: where it all begins. Dev. Biol. 245, 237-254.

5 Sato, K., Iwasaki, T., Hirahara, S., Nishihira, Y., and Fukami, Y. (2004) Molecular dissection of egg fertilization signaling with the aid of tyrosine kinase-specific inhibitor and activator strategies. Biochim. Biophys. Acta 1697, 103-121.

6 Sato, K., Aoto, M., Mori, K., et al. (1996) Purification and characterization of a src-related p57 protein-tyrosine kinase from Xenopus oocytes. J. Biol. Chem. 271, 13,250-13,257.

7 Sato, K., Iwao, Y., Fujimura, T., et al. (1999) Evidence for the involvement of a Src-related tyrosine kinase in Xenopus egg activation. Dev. Biol. 209, 308-320.

8 Sato, K., Tokmakov, A. A., Iwasaki, T., and Fukami, Y. (2000) Tyrosine kinase-depen-dent activation of phospholipase Cy is required for calcium transient in Xenopus egg fertilization. Dev. Biol. 224, 453-469.

9 Sato, K., Ogawa, K., Iwasaki, T., Tokmakov, A. A., and Fukami, Y. (2001) Hydrogen peroxide induces Src family tyrosine kinase-dependent activation of Xenopus eggs. Dev. Growth Differ. 43, 55-72.

10 Sato, K., Iwasaki, T., Ogawa, K., Konishi, M., Tokmakov, A. A., and Fukami, Y. (2002) Low density detergent-insoluble membrane of Xenopus eggs: subcellular microdomain for tyrosine kinase signaling in fertilization. Development 129, 885-896.

11 Murray, A. W. (1991) Cell cycle extracts. Methods Cell Biol. 36, 581-605.

12 Pappin, D. J. C., Hojrup, P., and Bleasby, A. J. (1993) Rapid identification of proteins by peptide-mass fingerprinting. Curr. Biol. 3, 327-332

13 James, P., Quadroni, M., Carafoli, E., and Gonnet, G. (1994) Protein identification in DNA databases by peptide mass fingerprinting. Protein Sci. 3, 1347-1350.

14 Eng, J. K., McCormack, A. L., and Yates, J. R., III (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976-989.

15 Yates, J. R., III, Eng, J. K., and McCormack, A. L. (1995) Mining genomes: correlating tandem mass spectra of modified and unmodified peptides to sequences in nucleotide databases. Anal. Chem. 67, 3202-3210.

16 Neubauer, G., King, A., Rappsilber, J., et al. (1998) Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex. Nat. Genet. 20, 46-50.

17 Choudhary, J. S., Blackstock, W. P., Creasy, D. M., and Cottrell, J. S. (2001) Interrogating the human genome using uninterpreted mass spectrometry data. Proteomics 1, 651-667.

18 Tokmakov, A. A., Sato, K., Iwasaki, T., and Fukami, Y. (2002) Src kinase induces calcium release in Xenopus egg extracts via PLCy and IP3-dependent mechanism. Cell Calcium 32, 11-20.

19 Sato, K., Tokmakov, A. A., He, C.-L., et al. (2003) Reconstitution of Src-dependent PLCy phosphorylation and transient calcium release by using membrane rafts and cell-free extracts from Xenopus eggs. J. Biol. Chem. 278, 38,413-38,420.

20 Sakakibara, K., Sato, K., Yoshino, K., et al. (2005) Molecular identification and characterization of Xenopus egg uroplakin III, an egg raft-associated transmembrane protein that is tyrosine-phosphorylated upon fertilization. J. Biol. Chem. 280, 15,029-15,037.

21 Mahbub Hasan, A. K. M., Sato, K., Sakakibara, K., et al. (2005) Uroplakin III, a novel Src substrate in Xenopus egg rafts, is a target for sperm protease essential for fertilization. Dev. Biol. in press.

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