Proteomic Profiling of Early Life Stages of European Grayling (

Proteomic Profiling of Early Life Stages of European Grayling (Thymallus thymallus) Spiros Papakostas,† L. Asbjørn Vøllestad,‡ Craig R. Primmer,† and Erica H. Leder*,† Division of Genetics and Physiology, Department of Biology, University of Turku, Finland, and Centre for Ecological and Evolutionary Synthesis, Department of Biology, University of Oslo, Norway Received May 20, 2010

Salmonids are teleost fish of profound research and economic interest. Embryonic development is a key aspect of salmonid biology that can be critically affected by environmental parameters. Still, their proteome during embryogenesis remains largely unexplored. This study investigates the proteome of the eyed-egg and hatching stages of embryonic development of a salmonid species, European grayling (Thymallus thymallus), using a shotgun proteomic approach. To deal with limited grayling protein resources, the generated spectra were compared against an all-salmonid database using search and multiple protein grouping algorithms to infer identifications. Functional enrichment analysis was carried out at different levels (gene ontologies, pathways, networks) using zebrafish as a reference genome. A total of 213 and 239 proteins were confidently detected in eyed and hatching stages, respectively. Cell cycle, energy, and protein metabolism were the major processes common to both stages. Nuclear activity and brain and eye development were the predominant functions in the eyed-stage proteome, while central nervous system, skeletal muscle, and heart development prevailed in the hatching stage. Overall, this research constitutes the first effort to describe the proteome during embryogenesis in grayling or any salmonid species. It also presents a systematic approach by which existing resources can enable proteome research in salmonids. Keywords: teleosts • salmonids • grayling • embryonic development • mass spectrometry

Introduction Teleost fishes are the largest and most variable vertebrate taxon.1 With about 27 000 recorded species, they comprise more than 99% of all known ray-finned fish (actinopterygians), which in turn include more than 95% of all living fish.2 Their pronounced diversity is reflected in multiple aspects of their biology such as morphology, ecology, behavior, genome organization, and development.1,2 Of particular interest is embryonic development in oviparous fishes since it can be profoundly affected by a number of environmental factors such as water temperature and dissolved oxygen concentrations.3 Adaptation to environmental conditions has been suggested to be of great importance during egg incubation and the critical phase that follows fry emergence in teleost ectotherms.4,5 At the molecular level, fast-evolving cis-regulatory and protein coding DNA sequences are considered to play an important role in teleost diversity.6 More generally, Carroll7 highlighted the evolutionary significance of such molecular level phenomena, especially when they affect proteins important for embryogenesis. In this framework, studying the embryonic proteome of teleost fish can be of fundamental and applied interest. * To whom correspondence should be addressed. Mailing address: Vesilinnantie 5, 20014 Turku, Finland. Phone: +358 2 333 7086. Fax: +358 2 333 6680. E-mail: [email protected]. † University of Turku. ‡ University of Oslo.

4790 Journal of Proteome Research 2010, 9, 4790–4800 Published on Web 07/05/2010

Recent advances in mass spectrometry (MS)-based proteomics have simplified large scale identification and quantitative profiling of organismal proteomes.8 Similarly, the ever-increasing amount of information on protein and protein-protein interactions allows for genes to be systematically reviewed and classified according to their function(s), involvement in particular process(es) or subcellular location(s) into annotated functional categories, pathways, and networks.9,10 This information constitutes a significant resource for functional proteomic studies especially when studying species for which only limited molecular information is available.11,12 To date, the proteome of the teleost embryo has been investigated in very few species.13 In fact, most studies have focused on a single species, the zebrafish (Danio rerio),13-16 which has been extensively employed as an experimental model for embryogenesis, organogenesis, and general development in vertebrates.17 Clearly, proteomic studies of a more diverse group of teleost species are required in order to better understand the molecular basis of teleost embryonic development. Salmonid fishes have been the focus of considerable research due to their economic importance, as well as their ecological and evolutionary uniqueness.18 Particularly, the evolutionary importance of early life history traits has been repeatedly emphasized because they can affect juvenile competitive ability, dispersal, foraging, and vulnerability to predation and climatic conditions.4,19-23 However, the molecular basis underlying ecologically relevant early life history traits has rarely, if ever, 10.1021/pr100507s

 2010 American Chemical Society

Proteomic Profiling of Early Life Stages of European Grayling been studied in any salmonid fish. In European grayling (Thymallus thymallus), the evolutionary importance of embryonic development rate and hatching time has been demonstrated in recently introduced populations inhabiting the Lesjaskogsvatnet lake system in southern Norway.24,25 Therefore, grayling individuals from this system can be useful candidates for investigating early development at the protein expression level in the context of evolutionary and ecological adaptation. In this study, a MS-based approach was employed to identify similarities and differences of the proteomes expressed at the eyed-egg and hatching larvae stages in grayling from Lake Lesjaskogsvatnet. Overall, this work provides the first systematic investigation of the early development proteome in salmonids. Due to the limited number of available protein sequences in grayling, this study also demonstrates a way by which existing sequence resources can be used for the MSbased protein detection in any salmonid species.

Materials and Methods Sample Collection and Experimental Design. The samples derived from two grayling spawning sites (Sandbekken and Valåe) of the Lesjaskogsvatn lake system. They were the product of the artificial fertilization between the pooled gametes of 30 individuals (15 sires, 15 dams) per spawning population. Eggs from each population were reared in common garden conditions at 6 °C. Embryos were sampled daily from fertilization to when the yolk sac was absorbed. Individuals were selected from a time point corresponding to the appearance of eye pigmentation (98-100 degree days, eyed-egg stage) and from when more than 50% of the individuals are hatched (188 degree days, hatching stage). Four individuals from each population for each time point were used for protein analysis. All fish embryos used in this study were collected according to animal experimentation guidelines at the sampling location in Norway. Sample Preparation. Proteins were isolated from whole embryos using Tri Reagent (Sigma), following the manufacturer instructions. Protein pellets resulting from the extraction protocol were dissolved in 1% SDS and quantified using a DC protein assay (Bio-Rad). Because within-stage quantification will be subsequently investigated, samples that were used in this study for the MS analysis were labeled with iTRAQ reagents following the manufacturer’s protocol (Applied Biosystems). Briefly, 100 µg of each sample was acetone precipitated and resuspended in 20 µL of 0.5 M triethyl ammonium bicarbonate (TEAB) and 1 µL of 2% SDS (supplied in the iTRAQ kit). Samples were reduced by the addition of 2 µL of 50 mM tris-carboxyethyl phosphine hydrochloride (TCEP) and incubation for 1 h at 60 °C, and then alkylated by the addition of 1 µL of 200 mM methyl methane thiosulfonate (MMTS) and incubation for 10 min at room temperature (RT). Following this step, 10 µL of trypsin (1 µg/µL) (Promega, Madison, WI) was added, and samples were digested overnight at 37 °C. Ethanol was added to the iTRAQ labels, and the reconstituted labels were added to their respective samples. Samples were incubated for 1 h, combined into their respective multiplexes, and then evaporated. Samples were dissolved in 50 µL of 0.1% trifluoroacetic acid (TFA) and desalted using MacroSpin C18 columns (The Nest Group, Southborough, MA) according to the manufacturer’s directions. Samples were dissolved in isoelectric focusing buffer consisting of 2 M urea and 2% IPG buffer, pH 3-10 (GE Healthcare), and fractionated before mass spectrometry analysis using isoelectric focusing on a 13 cm, pH 3-10 linear, Immobiline DryStrip (GE Health Care). The strips were rehy-

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drated for 12 h and focused on an IPGphor (Amersham Pharmacia Biotech, Piscataway, NJ) using the following program: hold at 500 V for 1 h, linear gradient from 500 to 1000 V for 15 min, hold at 1000 V for 1 h, linear gradient from 1000 to 8000 V for 30 min, hold at 8000 V for 3.5 h. The gel strip was cut into six fractions and peptides were extracted from the gel strip by washing three times: first in 0.1% TFA, second in 0.1% TFA, 50% acetonitrile (ACN), and third in 0.1% TFA, 100% ACN, all for 15 min at 37 °C. The solution from the three washes was combined and evaporated. The peptides were dissolved in 0.1% TFA, 5% ACN and desalted using UltraMicroSpin C18 columns (The Nest Group) according to the manufacturer’s directions. Mass Spectrometry Analysis. LC-MS/MS analyses were made using a system consisting of a Waters Cap-LC (Waters, Milford, MA) coupled to a QSTAR Pulsar ESI-hybrid quadrupole time-of-flight instrument (Applied Biosystems, Foster City, CA). A 0.3 mm ×5 mm PepMap C18 µ-precolumn (LC Packings, Dionex, Sunnyvale, CA) was used for sample loading and was coupled to a 15 cm × 75 µm i.d. fused silica capillary column packed with 5 µm Magic C18 (Michrom BioResources, Inc., Auburn, CA) for separation. A 90 min binary gradient from 2% to 30% phase A was used at a flow rate of 200 µL/min (phase A ) 5% ACN, 0.1% HCOOH; phase B ) 95% ACN, 0.1% HCOOH). Data acquisition and instrument control was performed using Analyst QS 1.1 software (Applied Biosystems, Foster City, CA), with the mass spectrometer set to perform 1 s survey scans followed by two 2 s MS/MS scans of the two most intense peaks (10 cps), with dynamic exclusion for 5 min. Database Search and Protein Detection Parameters. ProteinPilot software (v.3, Applied Biosystems, ABI) was employed to infer protein identifications. The detected protein threshold in the Paragon algorithm was set to 1.3 (95% confidence level). The search database was comprised of all the salmonid proteins (14 885 in total) submitted in UniProt database (15.11 release). The common contaminants file provided by ABI (ABSciex_ContaminantDB file) was also appended to these protein sequences. The search effort was set to thorough ID, and both biological modifications and amino acid substitutions were included. The quality of the Paragon algorithm detection results was assessed by performing a false discovery rate (FDR) analysis at 1% global error rate using a decoy database as implemented within the proteomics system performance evaluation pipeline (PSEP) of the ProteinPilot software. The protein groups were created by the Pro Group algorithm of the ProteinPilot software. This algorithm helped reduce the protein redundancy of the search database since it groups together proteins with the same spectral evidence. Yet, these groups were further inspected on the basis of the multidetected protein groups suggested by the same algorithm. Multidetected groups that involved proteins annotated under the same gene name or name entries in UniProt were manually grouped together. For each protein, the number of credible spectra (g95% confidence) uniquely assigned to them was counted, and for proteins detected only in one of the stages, a one-tailed χ-square test with the Yates’ continuity correction was performed to examine whether the spectra present were significantly greater than zero (p e 0.05). Gene Ontology (GO) Enrichment Analysis. Hierarchical GO overrepresentation tests were performed in Cytoscape 2.6.326 using the BiNGO 2.3 plugin.27 Four gene lists were investigated. These involved all proteins that were detected in the eyed stage (gene set a), all those detected in the hatching stage (set b), those detected only in the eyed stage (set c), and those detected Journal of Proteome Research • Vol. 9, No. 9, 2010 4791

research articles only in the hatching stage (set d). The first two lists were used to investigate the general functional properties of each stage, whereas the two latter ones highlighted the functional differences between them. Since zebrafish is the most widely used teleost species in developmental studies and has a wellannotated genome, the D. rerio genome (Ensembl Zv8) was employed as a gene reference set for the overrepresentation tests. The D. rerio gene orthologs for the grayling proteins were found using the BLAST tool in the UniProt database, whereas the ontology files required for the interpretation and the structured visualization of the overrepresented GO terms were retrieved from the GO database (as of date 07-JAN-2010). The hypergeometric overrepresentation tests were performed at 0.05 level of significance with the Benjamini-Hochberg FDR multiple-testing correction.28 Functional relatedness of the overrepresented GO terms was inferred using the ClueGO Cytoscape plugin.29 While BiNGO reconstructs the hierarchical ontology trees, ClueGO uses kappa statistics to create networks of functional GO clusters.29 As above, the D. rerio genome was employed as a reference set, and hypergeometric enrichment tests with Benjamini-Hochberg correction were performed. The networks were generated by setting the specificity to medium and the connectivity to equal or greater than 0.1.29 The networks were visualized with the GOlorize tool.30 Due to term interdependency in the GO hierarchy, numerous GO terms of the same branch can be significantly overrepresented.27 Therefore, both high-level (more general) and low-level/terminal (more specific) GO terms were used to describe each group. Pathway and Network Analysis. Pathway and network analysis were performed with the Ingenuity Pathway Analysis (IPA) software, v.8.0. The Agilent zebrafish V2 microarray, which fairly represents the whole zebrafish genome, was selected as a reference set. Fisher exact tests were used to examine the significance (p e 0.05) of the different pathways and networks in the investigated gene lists. Since the Ingenuity Knowledge Base is built on human (Homo sapiens), mouse (Mus musculus), and rat (Rattus norvegicus) resources, pathways and network functions that had to do with cancer, diseases, toxicity, pathogen-influenced signals, and xenobiotic signals were not considered in the analyses.

Results and Discussion Proteins Detected. A total of 313 proteins were detected, 213 and 239 in the eyed and hatching stages, respectively, of which 139 were common to both stages (Supplementary Tables 1 and 2, Supporting Information). In addition to the minimum 95% confidence threshold and the maximum 1% FDR, the confidence of the reported detections can be assessed by the fact that on average, at least three credible peptides (g95% confidence) were assigned to each protein, namely, 3.1 and 4.3 for the eyed and the hatching stage, respectively. These numbers are without considering vitellogenins, the major components of the egg yolk, which were identified by an average of more than 100 credible peptides in each stage. The inclusion of vitellogenins to these calculations would have caused an upward bias of the average peptide numbers. Although a number of proteins (65 eyed stage and 87 hatching stage) were detected by a single credible peptide, these detections are still highly confident, since almost all these proteins had an unused score of greater than or equal to two, which corresponds to 99% detection confidence (Supplementary Table 1, Supporting Information). 4792

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Papakostas et al. Interestingly, the reported detections for the grayling spectra were derived from a salmonid search database that included less than 80 grayling proteins in total (data not shown). On top of this, only three grayling entries were among the 313 reported proteins (Supplementary Table 1, Supporting Information). Thus, almost all of the detections in grayling were based on non-grayling protein sequence information. Given that Thymallinae is one of the evolutionary most distant subfamilies within salmonid phylogeny,31,32 it is likely that the salmonid protein sequences can effectively be used together as a search database for MS-based protein detections in any salmonid species, following the procedure presented here. The ProGroup algorithm can efficiently group together most of the redundant protein entries. However, in a few cases, manual classification can be necessary, based on the reported multidetected protein groups (Supplementary Table 1, Supporting Information). Functions in Common. Twenty functionally related groups of GO terms were identified in both stages (Figure 1). Significant terms, at p e 0.05, were detected in 17 of these groups, and 12 were practically the same in the two stages (Table 1, Supplementary Tables 3 and 4, Supporting Information). These common groups were largely involved in functions concerning (a) protein synthesis and metabolism (translational elongation, translation elongation factor activity, ribosomal subunits, protein folding), (b) carbohydrate metabolism and energy production (glycolysis, L-malate dehydrogenase activity, protontransporting ATP synthase complex), and (c) DNA packaging and cell cycle regulation (nucleosome assembly, nucleosome, regulation of cell cycle) (Table 1). Protein complexes concerning cell cycle, protein synthesis, and energy metabolism have been found to occupy central (core) positions in the yeast proteome network,33 with the complexes themselves consisting of core, module, and attachment proteins.34,35 In contrast to module and attachment proteins, core ones were almost always present in the complex.34 In zebrafish, using two-dimensional gel electrophoresis to profile protein expression during embryonic development, a number of constant gel spots were noticed throughout developmental stages.14 An interesting hypothesis follows from these findings, namely, that the GO-based functional groups and proteins in common between the stages are part of the predominant grayling core interactome during embryonic development. However, more research is needed before any clear conclusions can be drawn. Evidence of pronounced activity involved in carbohydrate metabolism in both the eyed and hatching stages was found in the pathway analysis level as well. Nine out of 19 significantly detected pathways were in common between the two stages, out of which most of the metabolic pathways concerned carbohydrate metabolism (Table 2, Supplementary Tables 5 and 6, Supporting Information). Carbohydrate metabolism has been suggested to play a critical role in fish embryonic development, with glucose serving primarily as an energy compound but also as a substrate for the synthesis of both nucleic acids and polysaccharides.36 Other pathways detected in both stages involved the ILK, granzyme A, protein ubiquitination, and regulation of actin-based motility by Rho pathways (Table 2). The integrin-linked kinase (ILK) pathway is a highly conserved functional complex from invertebrates to mammals with a crucial role in embryonic development.37 It has been implicated in cell proliferation, adhesion, migration, differentiation, and brain and muscle development.37-39 There is also evidence that integrins regulate cell spreading and migration

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Proteomic Profiling of Early Life Stages of European Grayling

Figure 1. Combined network view of the GO-based functional groups in the grayling eyed and hatching stages. A total of 20 groups were detected. Dashed lines distinguish the generated clusters to the biological process, cellular component, or molecular functions GO categories. For each category, every group has its own distinctive color. Multicolored nodes represent GO terms that belong to more than one group. A description of each group on the basis of the high- (more general) and low-level significant GO terms is given in Table 1. Since this is a combined view of the eyed and hatching stage networks, nodes were set to the same size, while node abundances and connectivity are only indicative of the general network organization. More details can be found in Supplementary Tables 3 and 4, Supporting Information. Table 1. Description of the GO-Based Functional Groups Identified by ClueGO in the Eyed (eye) and Hatching (hatch) Stagesa functional group

stage detected

high-level GO

terminal GO

Biological Process G1 G2 G3 G4 G5 G6 G7 G8 G9 G10

cellular homeostasis cellular macromolecule metabolic process macromolecule biosynthetic process DNA metabolic process cellular macromolecular complex subunit organization protein metabolic process carbohydrate catabolic process lipid localization ns nucleobase, nucleoside, and nucleotide metabolic process

eye both both eye both both both both

cellular iron-ion homeostasis regulation of cell cycle translational elongation DNA replication initiation nucleosome assembly protein folding glycolysis lipid transport ns nucleotide metabolic process

G11 G12 G13 G14 G15

Cellular Component intracellular non-membrane-bound organelle both mitochondrial membrane part both chromosomal part both ribonucleoprotein complex both proteasome complex hatch

keratin filament proton-transporting ATP synthase complex nucleosome large/small ribosomal subunit proteasome core complex

G16 G17 G18 G19 G20

Molecular Function ns hydrolase activity, acting on acid anhydrites ns oxidoreductase activity, acting on CH-OH group of donors translation factor activity, nucleic acid binding

ns nucleoside triphosphatase activity ns L-malate dehydrogenase activity translation elongation factor activity

eye

eye both both

a The most significant high-level terms derived from ClueGO, whereas the low-level, terminal ones derived from BiNGO. Three of the groups (G9, G16, and G18) had no significant terms (ns) at p e 0.05. All the significant GO terms for the BiNGO and ClueGO analyses are given in Supplementary Tables 3 and 4, Supporting Information, respectively.

through activation of Rho pathways.39 Lastly, the granzyme A pathway is involved in apoptosis,40 while the protein ubiquitination pathway is part of the protein catabolic mechanism. The presence of these pathways in both proteomes underlines the organogenesis-related processes of cell proliferation, migration, and death among their common

functions. Previous studies have reported a number of proteins associated with integrin and apoptosis signaling in hatching stage zebrafish embryos.16 According to the results of this work, the functioning of these pathways is likely to be present earlier in development and presumably throughout teleost embryogenesis. Journal of Proteome Research • Vol. 9, No. 9, 2010 4793

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Table 2. The Pathways That Were Found To Be Significantly Present (p e 0.05; in Bold When p e 0.01) in the Eyed (eye) and Hatching (hatch) Stagesa p-value

Table 3. The Top Five Networks Identified by the IPA Software and Their Five Most Significant Functions in the Eyed (NE1-NE5) and Hatching (NH1-NH5) Stagesa name

network functions

p value

protein synthesis molecular transport nucleic acid metabolism small molecule biochemistry cellular assembly and organization cellular assembly and organization hepatic system development and function cell death RNA post-transcriptional modification cell-to-cell signaling and interaction molecular transport cell-to-cell signaling and interaction lipid metabolism cardiovascular system development and function visual system development and function lipid metabolism small molecule biochemistry carbohydrate metabolism molecular transport cell morphology protein synthesis molecular transport nucleic acid metabolism small molecule biochemistry cell cycle protein synthesis cellular assembly and organization hepatic system development and function cell-to-cell signaling and interaction cellular function and maintenance protein synthesis post-translational modification protein folding molecular transport nucleic acid metabolism skeletal and muscular system development and function tissue morphology cell death cell morphology gene expression lipid metabolism small molecule biochemistry vitamin and mineral metabolism molecular transport carbohydrate metabolism protein synthesis cell death cellular assembly and organization cellular function and maintenance DNA replication, recombination and repair

2.00 × 10-8 6.91 × 10-4 6.91 × 10-4 6.91 × 10-4 3.36 × 10-3 3.44 × 10-5 4.01 × 10-5

eye

hatch

NE1

Amino Acid Metabolism arginine and proline metabolism glutathione metabolism phenylalanine, tyrosine, tryptophan biosynthesis

b 0.033 0.033

0.014 b 0.035

NE2

Carbohydrate Metabolism citrate cycle fructose and mannose metabolism glycolysis/gluconeogenesis glyoxylate and dicarboxylate metabolism pentose phosphate pathway pyruvate metabolism

0.012 0.033 0.000 0.037 0.012 0.002

0.013 b 0.001 0.040 b 0.014

b

0.016

0.000

b

NE4

Cellular Growth, Proliferation and Development ILK signaling 0.011 RAN signaling 0.002

0.014 b

NE5

Cellular Immune Response granzyme A signaling

0.000

0.001

Intracellular and Second Messenger Signaling Calcium Signaling b Protein Ubiquitination Pathway 0.004 RhoA Signaling b

0.010 0.018 0.004

pathway name

Metabolic Pathways

NE3

Energy Metabolism methane metabolism Nucleotide Metabolism purine metabolism Signaling Pathways

NH1

Neurotransmitters and Other Nervous Systems Signaling regulation of actin-based motility by Rho 0.036 0.002 Organismal Growth and Development actin cytoskeleton signaling b

NH2 0.009

a The genes mapped onto the Agilent zebrafish V2 array platform are given in Supplementary Table 5. The genes involved in each of the significant pathways can be found in Supplementary Table 6. b Pathways not significantly detected.

Last, many network functions were the same between the two stages as well (Table 3, Supplementary Table 7, Supporting Information). As with GO functional categories and pathways, the identified proteins grouped into networks that were involved with macromolecule/energy metabolism (carbohydrate, nucleic acid and lipid metabolism, protein synthesis), in apoptosis (cell death), and in cell proliferation mechanisms (cellular assembly and organization), but also an additional category in the network analysis included developmental processes (hepatic system development and function) (Table 3). These functions provide a general overview of the underlying commonalities between the two proteomes. Future studies, investigating more development stages, will likely reveal whether these common functions exist throughout embryonic development. Eyed-Stage Specific Functions. Brain and eye development were the predominant functions within the eyed stage. Based on spectra counts, 13 proteins were found to be significantly present only in the eyed stage (Supplementary Table 8, Supporting Information). Among them, the myristoylated alaninerich C-kinase substrate (MARCKS) and the epsilon isoform of 14-3-3 proteins have been known to play a critical role in brain 4794


NH3

NH4

NH5

2.63 × 10-4 3.05 × 10-4 5.11 × 10-4 2.31 × 10-4 2.91 × 10-4 5.97 × 10-4 6.91 × 10-4 6.91 × 10-4 5.53 × 10-4 5.53 × 10-4 1.07 × 10-3 1.07 × 10-3 1.35 × 10-3 3.43 × 10-6 2.02 × 10-3 2.02 × 10-3 2.02 × 10-3 2.70 × 10-3 5.46 × 10-15 3.44 × 10-5 4.01 × 10-5 5.11 × 10-4 5.79 × 10-4 3.66 × 10-5 4.90 × 10-4 4.90 × 10-4 6.91 × 10-4 6.91 × 10-4 1.77 × 10-4 1.77 × 10-4 2.63 × 10-4 1.35 × 10-3 2.91 × 10-3 4.80 × 10-6 4.80 × 10-6 4.80 × 10-6 9.28 × 10-6 2.78 × 10-5 1.81 × 10-5 1.60 × 10-4 1.90 × 10-4 1.90 × 10-4 4.78 × 10-4

a Details concerning the molecules that participate in each of these networks can be found in Supplementary Table 7, Supporting Information.

and eye development. In neonatal mouse brains, MARCKS and MARCKS-like proteins were highly expressed in the retina of the developing eye,41 regulating retinal cell proliferation.42 MARCKS-deficient mice displayed lethal brain deficiencies


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Figure 2. (A) Detailed view of the molecule interactions for the network NE3 identified in the eyed stage. Its functions include “cardiovascular and visual system development”, which to some extent characterizes the specific functioning of the eyed-stage proteome. (B) Detailed view of the hatching-stage network NH3, which is involved in “skeletal and muscular system development and function”. More information about the interacting molecules is given in Supplementary Table 7, Supporting Information. Both networks were drawn in Cytoscape 2.6.3 according to the results generated by the IPA software.

including lamination abnormalities of the cortex and the eye retina.43 Similarly, the 14-3-3 protein has been implicated in the eye and the early neural development in Drosophila and mice.44,45 On a different functional level, five of the detected pathways were also significant only in the eyed stage (Table

2). Again, the RAN pathway is involved in neural, brain and eye development. During mouse embryogenesis Ran protein expression was detected initially in neuroectoderm, neural groove, neural folds, neural tube, and later within the encephalic vesicle wall and spinal cord, especially during eye developJournal of Proteome Research • Vol. 9, No. 9, 2010 4795

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ment, including optic vesicle. In Drosophila embryos, Ran expression was restricted to central nervous system neuroblasts and was implicated in the eye/antenna disk development,47 whereas during Xenopus embryogenesis, Ran expression was again localized in the central nervous system as well as in neural crest, mesenchyme, eyes, and otic vesicles.48 Lastly, at the network functional level, one of the significant functions of the eyed-stage detected network, NE3, is “visual system development and function” (Table 3). A number of genes involved in the NE3 network concerned proteins detected only in the eyed stage (Figure 2A). Complement-controlled organogenesis processes were also among the predominant properties of the eyed-stage proteome. Complement component C3 was significantly detected only in the eyed stage (Supplementary Table 8, Supporting Information). Complement component 1Q and a serine proteinase inhibitor (serpin), the alpha-1 antiprotease-like protein, were also detected only in the eyed stage (Supplementary Table 8, Supporting Information). Serpins are proteins that, among other functions, control complement activation, programmed cell death, and development.49 Complement-regulated apoptotic pathways can have an important role in a number of early development and cell differentiation processes.50 In fact, neurons of the inner nuclear layer of the vertebrate eye retina undergo considerable cell death during early embryonic stages.51 Lange et al.52 detected C3 transcripts throughout the embryonic development of Atlantic cod in a number of tissues, including the eye. In general, apoptosis and phagocytosis can be pivotal to organogenesis as well as to the establishment of the nervous and immune systems during the early stages of development.52,53 Last, the comparison of the two stages at the GO level revealed, among other things, that terms related to nuclear activity (DNA replication, nuclear transport) were particularly overrepresented in the proteins detected at the eyed stage (Table 4, Figure 3). In agreement with this, at the pathway level, the purine metabolism pathway was significant only in the eyed stage (Table 2). Furthermore, and in contrast to the apparent similarity of the most significant network functions (Table 3), in many cases different underlying processes were predominant for each stage, and under the “cell morphology” network function, the most significant mechanism in the eyed stage was “morphology of nuclear matrix”, while in the hatching stage it was “morphology of microfilaments” (results not shown). It is likely that the increased nuclear activity at the eyed stage is directly connected to the increased cell division required for embryonic growth and organogenesis during early embryogenesis.54 In teleost species, little is known about the proteome functioning during the eye pigmentation of the embryo, yet such early development stages can critically affect the whole embryogenesis and adult growth processes.5 In zebrafish, eye pigmentation becomes apparent around 24 h postfertilization (hpf) at the beginning of the pharyngula stage.55 By twodimensional gel electrophoresis, significant changes in the protein profile were found to accompany the zebrafish embryo entering the pharyngula development period.14 From use of MS on gel spots corresponding to abundant or stage-specific proteins, a number of cytosolic, cytoskeletal, and nuclear protein identifications, many of which were also detected in grayling, were reported.14 Based on these identifications, processes involved in metabolism, cytoskeleton, translation, and protein degradation have been suggested to be present in the 24 4796


Table 4. All the Terminal Overrepresented GO Categories Found in Gene Set c (Genes Derived from Proteins That Were Detected Only in the Eyed Stage) and in Set d (Proteins Detected Only in Hatching Stage)a corrected p-value GO term

eye (set c)

Biological Process DNA replication initiation 7.60 × 10-4 heterocycle metabolic process 4.79 × 10-2 negative regulation of coagulation nucleoside metabolic process 1.85 × 10-2 response to hypoxia ribonucleoside monophosphate 3.47 × 10-2 biosynthetic process RNA interference 3.88 × 10-2 translational elongation ubiquitin-dependent protein catabolic process Cellular Component cytosol 1.45 × 10-2 integral to mitochondrial inner 3.88 × 10-2 membrane PCNA complex proteasome core complex ribonucleoside-diphosphate 3.88 × 10-2 reductase complex ribosome RNA-induced silencing complex 3.88 × 10-2 Molecular Function betaine-homocysteine S-methyltransferase activity calcium-dependent phospholipid binding calcium ion binding creatine kinase activity cytochrome c oxidase activity DNA polymerase processivity factor activity IMP cyclohydrolase activity 3.88 × 10-2 nucleoside triphosphatase 5.89 × 10-3 activity orotate 3.88 × 10-2 phosphoribosyltransferase activity orotidine-5′-phosphate 3.88 × 10-2 decarboxylase activity phospholipase inhibitor activity phosphoribosylaminoinidazole3.88 × 10-2 carboxamide formyltransferase activity ribosome binding structural constituent of ribosome threonine-type endopeptidase activity transaldolase activity transketolase activity 3.88 × 10-2

hatch (set d)

2.12 × 10-2 4.57 × 10-2

3.67 × 10-2 4.94 × 10-2

4.57 × 10-2 3.27 × 10-3 4.38 × 10-21

4.57 × 10-2 4.00 × 10-2 2.17 × 10-2 4.57 × 10-2 4.57 × 10-2 4.57 × 10-2

1.89 × 10-2

2.25 × 10-3 3.61 × 10-22 3.27 × 10-3 4.57 × 10-2

a More details are given in Supplementary Table 3, Supporting Information.

hpf zebrafish embryo.14 By investigating more detected proteins and carrying out the functional analysis at different levels, the present work has additionally revealed that cell proliferation and cell death, as well as early neural, brain, and eye development functions are among the major functions of the eyed-stage proteome in grayling and likely in other teleosts as well.


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Figure 3. The result from the direct comparison of the eyed and hatching stages with ClueGO. Color gradient shows the proportion of genes in the GO term associated with each of the two stages. Red coloring characterizes eyed-stage related GOs, while green the hatching-stage ones. Equal proportions are shown in white. Node size is related to the statistical significance of the GO. Only terms with a corrected p-value g 0.05 are effectively visible. Edge line width is proportional to the kappa scores, while node connectivity was inferred using kappa statistics.22 Seven overrepresented GO terms and term clusters were related to the eyed stage (E1-E7) and eight to the hatching stage (H1-H8). The most significant GOs for the clusters or terms are nucleotide biosynthetic process (E1), iron-ion transport (E2), nuclear transport (E3), DNA replication initiation (E4), mitochondrial membrane part (E5), mitochondrial matrix (E6), ribose phosphate diphosphokinase activity (E7), NADPH regeneration (H1), regulation of actin filament polymerization (H2), protontransporting ATP synthase complex (H3), proteasome core complex, alpha subunit complex (H4), ribosome (H5), inorganic cation transmembrane transporter activity (H6), translation factor activity, nucleic acid binding (H7), threonine-peptidase activity (H8). More details can be found in Supplementary Table 4, Supporting Information.

Figure 4. Scatter plot of the average number of unique credible (g95 confidence) spectra per technical replicate assigned to each of the 313 detected proteins in the eyed and hatching stages. The names of those proteins with the most assigned spectra are noted on the graph. A detailed view of these data can be found in Supplementary Table 8, Supporting Information.

Hatching-Specific Functions. A total of 19 proteins and five pathways were significantly detected only in the hatching stage (Table 2, Supplementary Table 8, Supporting Information). Skeletal muscle development was found to be a major hatching-specific function. Creatine kinases 1 and 2, both among the significant proteins, are involved in muscle-specific metabolism.56 Troponin T isoform 1, another significant protein, is involved in muscle maturation.57 In addition to the specific presence of particular proteins, their relative abundance, as can be inferred by the spectral evidence,58,59 also highlights the muscle functioning of the hatching-stage detected proteins. A number of skeletal muscle proteins, namely the myosin heavy chain, actin alpha, and actin beta, had considerably higher spectral counts in the hatching than in the eyed stage (101, 80, and 103 versus 2.6, 23, and 8, respectively; Figure 4, Supplementary Table 8, Supporting Information). Evidence for hatching-specific myoskeletal development functioning can also be found in the GO, pathway, and network levels of

analysis. The GO terms of actin filament polymerization, creatine kinase activity, and calcium ion binding were significant only in the hatching stage (Figure 3, Table 4). The RhoA and actin cytoskeleton signaling pathways, both involved in muscle tissue development,60 were detected only in the hatching stage (Table 2). Lastly, one of the most significant functions of the network NH3 was “skeletal and muscular system development and function” (Table 3). A number of genes, whose proteins were detected only in the hatching stage, participate in this network (Figure 2B). Other hatching-specific functions involve central nervous system and heart development. In particular, the fatty acid binding protein (FABP) brain type and heart type are two proteins detected only in the hatching stage (Supplementary Table 8, Supporting Information). The FABP brain type is an intracellular protein expressed particularly in the central nervous system with an important role during neurogenesis in vertebrates.61,62 During zebrafish late embryogenesis, FABP Journal of Proteome Research • Vol. 9, No. 9, 2010 4797

research articles brain type mRNA transcripts were detected in the lateral floor plate of the spinal cord, the lateral hindbrain, the ventricular brain zone, the forebrain, and the retina.63 On the other hand, the FABP heart type has been involved in regulating cardiomyocyte growth and differentiation of neonatal hearts.64 Furthermore, in addition to taking part in the developmental processes of muscle system development, the Rho signaling pathways have been found to play an important role in neural system and heart development in zebrafish.65,66 RhoA has also been implicated in the heart development of chick embryos.67 Last, the comparison of the two stages at the GO level revealed terms related to (a) protein catabolism (ubiquitindependent catabolic process, proteasome core complex, threonine-peptidase activity), (b) hydrogen ion transmembrane transporter activity (cytochrome c oxidase activity), and (c) ribosome binding to be particularly overrepresented in the hatching-detected proteins (Table 4, Figure 3). Ubiquitinmediated proteolysis can have a critical role in both somite segmentation and differentiation processes by which the growing embryo will eventually develop its skeletal muscle, axial skeleton, and dermis.68 In agreement with these results, proteasome and ubiquitin up-regulation has been detected at both the transcriptome and the proteome level at later stages of zebrafish development.16,69 Previous efforts investigating the zebrafish hatching proteome (72 hpf) by employing two-dimensional gel electrophoresis, MS, and two-dimensional PAGE methodologies identified numbers of proteins largely associated with organ systems such as central nervous system, heart, and skeletal muscle.16 At the cellular level, these proteins were related to structure, transcription/translation, cell cycle, ion transport, and nucleotide, carbohydrate, lipid and energy metabolism.16 Pathways with the most contributing proteins were related to calcium, integrin, extracellular signal-regulated kinase/mitogen-activated protein kinase, and vascular endothelial growth factor signaling.16 These results are in good agreement with the findings of the present work, suggesting similar functioning of the hatching-stage proteome between zebrafish and grayling. This agreement additionally strengthens the validity of the methodology presented here, highlighting a way by which the salmonid embryonic proteome can be studied, even in species with limited genomic/proteomic information.

Conclusions This work is the first systematic investigation of the embryonic proteome functioning in European grayling and in salmonids in general. Based on the proteins detected, the analyses that were carried out at multiple levels (spectra counts, GOs, pathways, and networks) largely agreed but also complemented each other with respect to both the similarities and the differences of the two proteomes. The common functions had to do primarily with cell cycle, energy, and protein metabolism. In both stages, glycolytic and cell proliferation pathways/ networks highlighted the energy and growth requirements of the developing embryo. Cell migration, cell differentiation, and apoptotic processes were related to organogenesis within the two proteomes. Significant nuclear activity together with early neural, brain, and eye development-related pathways and network functions were predominant in the eyed-stage proteome. On the other hand, central nervous system, heart, and myoskeletal system developmental processes were among the functions that characterized the hatching-stage proteome. Given that MS-based proteomics deals with the detection of 4798


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the most abundant proteins, this study likely describes the major functions of the two proteomes. Yet, functional classification of the detected proteins could serve as a valuable resource for future efforts targeting the grayling or salmonid proteome.

Acknowledgment. This work has been financially supported by the Research Council of Norway and the Finnish Centre of Excellence in Genetics and Physiology. We would like to thank Thrond Haugen, Gaute Thomassen, and Nicola Barson for fieldwork assistance and sampling the grayling embryos, the Turku Centre for Biotechnology Proteomics Facility for technical assistance, particularly Anne Rokka and Arttu Heinonen, and Robert Molder for useful discussions concerning proteomic analyses. Supporting Information Available: Tables containing the ProteinPilot unused and total scores, percent coverage, number of assigned peptides and UniProt identifiers for the salmonid detected proteins; the names of the proteins detected in each of the eyed and hatching stages; the results of the BiNGO analysis for proteins detected in the eyed stage (set a), the hatching stage (set b), the unique to eyed stage (set c) and the unique to hatching stage (set d); the significant GO terms (p e 0.05) for the combined and the comparison GO-based functional grouping analysis performed with ClueGO; the zebrafish ortholog genes mapped onto the Agilent zebrafish V2 microarray by the IPA software for the eyed (set a) and the hatching (set b) stage; the zebrafish V2 microarray mapped genes associated with each of the significantly detected pathways by the IPA software in the eyed and the hatching stage; the zebrafish V2 microarray genes associated with the IPA top five detected networks in the eyed (networks NE1 to NE5) and hatching (NH1 to NH5) stages; and the unique (at g 95 confidence) spectra assigned to each of the reported proteins per pooled sample (iTRAQ run). This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Nelson, J. S. Fishes of the world, 4th ed.; John Wiley and Sons: New York, 2006. (2) Volff, J. N. Genome evolution and biodiversity in teleost fish. Heredity 2005, 94, 280–294. (3) Johnston, I. A. Environment and plasticity of myogenesis in teleost fish. J. Exp. Biol. 2006, 209, 2249–2264. (4) Jensen, L. F.; Hansen, M. M.; Pertoldi, C.; Holdensgaard, G.; Mensberg, K. L. D.; Loeschcke, V. Local adaptation in brown trout early life-history traits: implications for climate change adaptability. Proc. R. Soc. B: Biol. Sci. 2008, 275, 2859–2868. (5) Macqueen, D. J.; Robb, D. H. F.; Olsen, T.; Melstveit, L.; Paxton, C. G. M.; Johnston, I. A. Temperature until the ‘eyed stage’ of embryogenesis programmes the growth trajectory and muscle phenotype of adult Atlantic salmon. Biol. Lett. 2008, 4, 294–298. (6) Ravi, V.; Venkatesh, B. Rapidly evolving fish genomes and teleost diversity. Curr. Opin. Genet. Dev. 2008, 18, 544–550. (7) Carroll, S. B. Evo-devo and an expanding evolutionary synthesis: A genetic theory of morphological evolution. Cell 2008, 134, 25– 36. (8) Gstaiger, M.; Aebersold, R. Applying mass spectrometry-based proteomics to genetics, genomics and network biology. Nat. Rev. Genet. 2009, 10, 617–627. (9) Okuda, S.; Yamada, T.; Hamajima, M.; Itoh, M.; Katayama, T.; Bork, P.; Goto, S.; Kanehisa, M. KEGG Atlas mapping for global analysis of metabolic pathways. Nucleic Acids Res. 2008, 36, W423–W426. (10) Rhee, S. Y.; Wood, V.; Dolinski, K.; Draghici, S. Use and misuse of the gene ontology annotations. Nat. Rev. Genet. 2008, 9, 509–515. (11) Carpentier, S. C.; Panis, B.; Vertommen, A.; Swennen, R.; Sergeant, K.; Renaut, J.; Laukens, K.; Witters, E.; Samyn, B.; Devreese, B. Proteome analysis of non-model plants: A challenging but powerful approach. Mass Spectrom. Rev. 2008, 27, 354–377.

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