BACKGROUND
the zebrafish has become a widely used vertebrate model system for which a large tool-box of genetic and cell biological methods has been established  <cit> .
research using zebrafish is further supported by the zebrafish sequencing project, which has facilitated the generation of microarrays for large scale expression profiling.
it has been proposed that proteomics should complement the genome-wide expression profiling  <cit> .
however, a major obstacle in the application of proteomics has been the high proportion of yolk proteins in early embryos.
proteomic studies in zebrafish have therefore been limited to adult tissues  <cit> .
one study targeted larval stages  <dig> or  <dig> hpf , when the yolk to cell mass ratio is already decreased  <cit> , however, without identifying the proteins.
therefore, it remains unclear whether at this stage analysis without deyolking provides satisfactory information about cellular proteins.
thus, the development of a reliable method to remove the interfering yolk from cells on a large scale is required to apply proteomics to early embryos.
here, we provide detailed protocols for all zebrafish-specific steps of a proteomic experiment from dechorionation to mass spectrometry-based protein identification.
as a key step, we present and validate a method for batch removal of the yolk from early embryos.
RESULTS
deyolking of embryos
in the early embryo, the cells forming the embryo proper constitute only a minor volume of the embryo compared to the large yolk cell .
the abundance of yolk proteins interferes with any proteomic application that intends to target the cells of the embryo proper.
the major yolk protein vitellogenin, a phospholipo-glycoprotein, functions as a nutritional source for the developing embryo  <cit> .
figure 1a demonstrates how several isoforms and degradation products of vitellogenin obscure the 2d gel image completely.
to generate enhanced 2d gels from early embryos, we developed a method for rapid batch removal of the yolk.
the method takes advantage of the high mechanical instability of the big yolk cell compared to the smaller cells of the embryo proper.
by pipetting with a narrow tip, the yolk cell can be disrupted.
a buffer of low osmolarity facilitated the dissolving of the yolk.
the deyolking efficiency was further increased by two additional wash steps.
by removing the yolk proteins this method efficiently decreased the total protein amount per embryo more than  <dig> fold from  <dig> to  <dig> μg per embryo .
however, recovery of cellular proteins remained high as evident by only a minor reduction in signal intensity of tubulin and mek as detected by western blotting .
we assume that this minor decrease is due to the loss of the fraction of mek and tubulin that is expressed in the yolk and yolk syncytial layer .
there were no major changes in the efficiency of deyolking or the protein amount per embryo between high  and tailbud  stages.
western blotting
the success of western blotting depends on the affinity and specificity of the antibodies used and on the abundance of the target protein.
if the yolk is not removed manually, then only  <dig> or  <dig> embryos  can be loaded per lane on a gel to avoid overloading effects due to yolk protein.
this limits the sensitivity for cellular proteins.
the deyolking method enabled us to load significantly more embryos and therefore the signal from specific cellular proteins was increased.
the failure to detect ezrin/radixin/moesin without deyolking may be due to electrophoretic distortions caused by abundant yolk sac proteins.
loading only  <dig> embryo , the dominant yolk protein induced major distortions in the molecular weight range of  <dig> to  <dig> kd.
this decreased resolution and consequently also sensitivity around this mass range.
furthermore, the total protein amount that can be loaded without overloading artefacts is reduced in samples containing few, very abundant proteins compared to samples with uniformly distributed proteins, as after deyolking .
we assume therefore that the increase in sensitivity gained due to this deyolking method even exceeds what would be anticipated due to the  <dig> fold reduction in total protein.
2d gel electrophoresis
the establishment of the deyolking protocol was a prerequisite for high quality 2d gels from early zebrafish embryos.
after removal of the predominant yolk proteins we were able to generate high resolution 2d gels in the acidic  as well as in the basic range .
we established a protocol that is compatible with three colour fluorescent labelling using the ettan dige system , which significantly reduces inter-gel variability compared to one colour stains  <cit>  while the sensitivity is comparable to silver staining with a detection limit of less than  <dig> ng protein  <cit> .
mass spectrometry
identification of proteins by mass spectrometry, using conventional search algorithms, depends on the representation of the sequence  in the searched database.
since the genome sequencing project of zebrafish is still ongoing, sequence coverage in the databases is incomplete.
we therefore compared three databases in regard to the success rate in protein identification from zebrafish samples.
the number of zebrafish entries in ensembl, tigr, msdb and ncbinr database is presented in table  <dig>  msdb and ncbinr are two general databases commonly used for ms database search.
the zebrafish peptide database of ensembl is generated by automatic annotation of sequences generated by the zebrafish genome project  <cit> .
the tigr gene index integrates est-sequencing data from international zebrafish gene research projects and assembles single ests to "tentative consensus" sequences   <cit> .
we compared the identification rate of these databases by analysing  <dig> out of  <dig> spots automatically detected on a 2d gel.
besides a few higher expressed proteins, we selected many spots in the medium expression range or even close to the detection limit for ms-identification .
in-gel tryptic digests of protein spots were analysed by maldi-tof and the peptide mass fingerprints  were subsequently searched against ensembl, tigr and msdb databases .
all identifications were manually validated taking into account the mowse score, apparent versus predicted isoelectric point and molecular weight, as well as the error distribution of apparent versus predicted peptide masses .
in total,  <dig> out of  <dig> spots were successfully identified.
figure  <dig> demonstrates how the identifications are distributed among the databases.
<dig> proteins that were identified from several spots were included only once in the statistics to avoid a bias.
none of the databases alone was sufficient to identify all  <dig> unique spots.
using ensembl and tigr databases  <dig> and  <dig> spots could be identified, respectively, while the lower number of zebrafish entries in the msdb database was reflected by a lower identification rate .
the combination of two databases was sufficient for more than 95% of the identifications.
we therefore routinely perform searches on msdb and ensembl databases.
the tigr database serves as an additional resource for samples which otherwise cannot be identified despite high quality spectra.
both the ensembl as well as the tigr project gather a rich pool of information regarding the database entries, such as protein name, homologies, links to gene information and protein-domains.
these valuable resources can be accessed directly by the identifiers of the respective proteins.
discussion
our development of an efficient deyolking protocol for early embryos will influence many areas of zebrafish research.
most remarkably, it enabled us to undertake comparative proteomics of zebrafish embryos, an approach that has already proven its value in many other systems.
in particular, the application of the dige system will support sensitive and quantitative analysis of protein expression differences.
in addition to gel-based proteomics, the deyolking protocol also is crucial for gel free mass spectrometry-based proteomics.
there are numerous applications for quantitative proteomics in zebrafish: analysis of specific zebrafish mutants as well as the analysis of gain and loss of functions induced by rna or morpholino injections will provide a better molecular understanding of genes and their functions during development.
also, chemical inhibitors could be applied to elucidate downstream targets of specific signalling pathways.
finally, in the field of toxicology, proteomics will complement existing techniques like microarray analysis to reveal the mode of action of toxic substances.
this will be increasingly important given that zebrafish as a model system in toxicology is on the rise  <cit> .
in addition to its application in proteomics deyolking will be beneficial to other subjects such as microarray experiments targeting the gene expression pattern in the embryo proper.
we have already applied the protocol successfully to such an experiment .
therefore, the deyolking protocol will prove a valuable tool in zebrafish research including manifold applications in biochemistry and proteomics as well as other areas.
CONCLUSIONS
we have established methods for proteomics of early zebrafish embryos.
the simple but efficient protocol established for yolk removal enabled the generation of high quality 2d gels.
it should further prove valuable for several other applications for which the yolk is detrimental.
here, we demonstrated that western blotting is improved significantly using the deyolking method.
the evaluation of mass spectrometry-based database searches revealed that the combination of two publicly available databases yields a good identification rate.
therefore, as a fruit of the zebrafish genome project, mass spectrometry-based identification of zebrafish samples is now possible.
we hope that the developed methods will facilitate and stimulate the application of proteomics in zebrafish embryos to analyse fundamental developmental processes.

 the zebrafish has become a widely used vertebrate model system for which a large tool-box of genetic and cell biological methods has been established. research using zebrafish is further supported by the zebrafish sequencing project, which has facilitated the generation of microarrays for large scale expression profiling. it has been proposed that proteomics should complement the genome-wide expression profiling. however, a major obstacle in the application of proteomics has been the high proportion of yolk proteins in early embryos. proteomic studies in zebrafish have therefore been limited to adult tissues. one study targeted larval stages or hpf, when the yolk to cell mass ratio is already decreased, however, without identifying the proteins. therefore, it remains unclear whether at this stage analysis without deyolking provides satisfactory information about cellular proteins. thus, the development of a reliable method to remove the interfering yolk from cells on a large scale is required to apply proteomics to early embryos. here, we provide detailed protocols for all zebrafish-specific steps of a proteomic experiment from dechorionation to mass spectrometry-based protein identification. as a key step, we present and validate a method for batch removal of the yolk from early embryos.  deyolking of embryos in the early embryo, the cells forming the embryo proper constitute only a minor volume of the embryo compared to the large yolk cell. the abundance of yolk proteins interferes with any proteomic application that intends to target the cells of the embryo proper. the major yolk protein vitellogenin, a phospholipo-glycoprotein, functions as a nutritional source for the developing embryo. figure 1a demonstrates how several isoforms and degradation products of vitellogenin obscure the 2d gel image completely. to generate enhanced 2d gels from early embryos, we developed a method for rapid batch removal of the yolk. the method takes advantage of the high mechanical instability of the big yolk cell compared to the smaller cells of the embryo proper. by pipetting with a narrow tip, the yolk cell can be disrupted. a buffer of low osmolarity facilitated the dissolving of the yolk. the deyolking efficiency was further increased by two additional wash steps. by removing the yolk proteins this method efficiently decreased the total protein amount per embryo more than fold from to μg per embryo. however, recovery of cellular proteins remained high as evident by only a minor reduction in signal intensity of tubulin and mek as detected by western blotting. we assume that this minor decrease is due to the loss of the fraction of mek and tubulin that is expressed in the yolk and yolk syncytial layer. there were no major changes in the efficiency of deyolking or the protein amount per embryo between high and tailbud stages. western blotting the success of western blotting depends on the affinity and specificity of the antibodies used and on the abundance of the target protein. if the yolk is not removed manually, then only or embryos can be loaded per lane on a gel to avoid overloading effects due to yolk protein. this limits the sensitivity for cellular proteins. the deyolking method enabled us to load significantly more embryos and therefore the signal from specific cellular proteins was increased. the failure to detect ezrin/radixin/moesin without deyolking may be due to electrophoretic distortions caused by abundant yolk sac proteins. loading only embryo, the dominant yolk protein induced major distortions in the molecular weight range of to kd. this decreased resolution and consequently also sensitivity around this mass range. furthermore, the total protein amount that can be loaded without overloading artefacts is reduced in samples containing few, very abundant proteins compared to samples with uniformly distributed proteins, as after deyolking. we assume therefore that the increase in sensitivity gained due to this deyolking method even exceeds what would be anticipated due to the fold reduction in total protein. 2d gel electrophoresis the establishment of the deyolking protocol was a prerequisite for high quality 2d gels from early zebrafish embryos. after removal of the predominant yolk proteins we were able to generate high resolution 2d gels in the acidic as well as in the basic range. we established a protocol that is compatible with three colour fluorescent labelling using the ettan dige system, which significantly reduces inter-gel variability compared to one colour stains while the sensitivity is comparable to silver staining with a detection limit of less than ng protein. mass spectrometry identification of proteins by mass spectrometry, using conventional search algorithms, depends on the representation of the sequence in the searched database. since the genome sequencing project of zebrafish is still ongoing, sequence coverage in the databases is incomplete. we therefore compared three databases in regard to the success rate in protein identification from zebrafish samples. the number of zebrafish entries in ensembl, tigr, msdb and ncbinr database is presented in table msdb and ncbinr are two general databases commonly used for ms database search. the zebrafish peptide database of ensembl is generated by automatic annotation of sequences generated by the zebrafish genome project. the tigr gene index integrates est-sequencing data from international zebrafish gene research projects and assembles single ests to "tentative consensus" sequences. we compared the identification rate of these databases by analysing out of spots automatically detected on a 2d gel. besides a few higher expressed proteins, we selected many spots in the medium expression range or even close to the detection limit for ms-identification. in-gel tryptic digests of protein spots were analysed by maldi-tof and the peptide mass fingerprints were subsequently searched against ensembl, tigr and msdb databases. all identifications were manually validated taking into account the mowse score, apparent versus predicted isoelectric point and molecular weight, as well as the error distribution of apparent versus predicted peptide masses. in total, out of spots were successfully identified. figure demonstrates how the identifications are distributed among the databases. proteins that were identified from several spots were included only once in the statistics to avoid a bias. none of the databases alone was sufficient to identify all unique spots. using ensembl and tigr databases and spots could be identified, respectively, while the lower number of zebrafish entries in the msdb database was reflected by a lower identification rate. the combination of two databases was sufficient for more than 95% of the identifications. we therefore routinely perform searches on msdb and ensembl databases. the tigr database serves as an additional resource for samples which otherwise cannot be identified despite high quality spectra. both the ensembl as well as the tigr project gather a rich pool of information regarding the database entries, such as protein name, homologies, links to gene information and protein-domains. these valuable resources can be accessed directly by the identifiers of the respective proteins. discussion our development of an efficient deyolking protocol for early embryos will influence many areas of zebrafish research. most remarkably, it enabled us to undertake comparative proteomics of zebrafish embryos, an approach that has already proven its value in many other systems. in particular, the application of the dige system will support sensitive and quantitative analysis of protein expression differences. in addition to gel-based proteomics, the deyolking protocol also is crucial for gel free mass spectrometry-based proteomics. there are numerous applications for quantitative proteomics in zebrafish: analysis of specific zebrafish mutants as well as the analysis of gain and loss of functions induced by rna or morpholino injections will provide a better molecular understanding of genes and their functions during development. also, chemical inhibitors could be applied to elucidate downstream targets of specific signalling pathways. finally, in the field of toxicology, proteomics will complement existing techniques like microarray analysis to reveal the mode of action of toxic substances. this will be increasingly important given that zebrafish as a model system in toxicology is on the rise. in addition to its application in proteomics deyolking will be beneficial to other subjects such as microarray experiments targeting the gene expression pattern in the embryo proper. we have already applied the protocol successfully to such an experiment. therefore, the deyolking protocol will prove a valuable tool in zebrafish research including manifold applications in biochemistry and proteomics as well as other areas.  we have established methods for proteomics of early zebrafish embryos. the simple but efficient protocol established for yolk removal enabled the generation of high quality 2d gels. it should further prove valuable for several other applications for which the yolk is detrimental. here, we demonstrated that western blotting is improved significantly using the deyolking method. the evaluation of mass spectrometry-based database searches revealed that the combination of two publicly available databases yields a good identification rate. therefore, as a fruit of the zebrafish genome project, mass spectrometry-based identification of zebrafish samples is now possible. we hope that the developed methods will facilitate and stimulate the application of proteomics in zebrafish embryos to analyse fundamental developmental processes. 
as a prerequisite to carry out proteomic experiments with early zebrafish embryos, we developed a method to efficiently remove the yolk from large batches of embryos. the provided protocols for proteomic analysis of early embryos enable research to be taken in novel directions in embryogenesis. while genetic methods are readily available in zebrafish, protocols for two dimensional gel electrophoresis and proteomics have yet to be developed. this method enabled high resolution 2d gel electrophoresis and improved western blotting considerably. here, we provide detailed protocols for proteomics in zebrafish from sample preparation to mass spectrometry, including a comparison of databases for ms identification of zebrafish proteins. zebrafish has become a powerful and widely used model system for the analysis of vertebrate embryogenesis and organ development.
 zebrafish has become a powerful and widely used model system for the analysis of vertebrate embryogenesis and organ development. while genetic methods are readily available in zebrafish, protocols for two dimensional gel electrophoresis and proteomics have yet to be developed.  as a prerequisite to carry out proteomic experiments with early zebrafish embryos, we developed a method to efficiently remove the yolk from large batches of embryos. this method enabled high resolution 2d gel electrophoresis and improved western blotting considerably. here, we provide detailed protocols for proteomics in zebrafish from sample preparation to mass spectrometry, including a comparison of databases for ms identification of zebrafish proteins.  the provided protocols for proteomic analysis of early embryos enable research to be taken in novel directions in embryogenesis. 