Quantitative Molecular Phenotyping of Gill Remodeling in a Cichlid Fish Responding to Salinity Stress
2013; Elsevier BV; Volume: 12; Issue: 12 Linguagem: Inglês
10.1074/mcp.m113.029827
ISSN1535-9484
AutoresDietmar Kültz, Johnathon Li, Alison M. Gardell, Romina Sacchi,
Tópico(s)Genetic diversity and population structure
ResumoA two-tiered label-free quantitative (LFQ) proteomics workflow was used to elucidate how salinity affects the molecular phenotype, i.e. proteome, of gills from a cichlid fish, the euryhaline tilapia (Oreochromis mossambicus). The workflow consists of initial global profiling of relative tryptic peptide abundances in treated versus control samples followed by targeted identification (by MS/MS) and quantitation (by chromatographic peak area integration) of validated peptides for each protein of interest. Fresh water acclimated tilapia were independently exposed in separate experiments to acute short-term (34 ppt) and gradual long-term (70 ppt, 90 ppt) salinity stress followed by molecular phenotyping of the gill proteome. The severity of salinity stress can be deduced with high technical reproducibility from the initial global label-free quantitative profiling step alone at both peptide and protein levels. However, an accurate regulation ratio can only be determined by targeted label-free quantitative profiling because not all peptides used for protein identification are also valid for quantitation. Of the three salinity challenges, gradual acclimation to 90 ppt has the most pronounced effect on gill molecular phenotype. Known salinity effects on tilapia gills, including an increase in the size and number of mitochondria-rich ionocytes, activities of specific ion transporters, and induction of specific molecular chaperones are reflected in the regulation of abundances of the corresponding proteins. Moreover, specific protein isoforms that are responsive to environmental salinity change are resolved and it is revealed that salinity effects on the mitochondrial proteome are nonuniform. Furthermore, protein NDRG1 has been identified as a novel key component of molecular phenotype restructuring during salinity-induced gill remodeling. In conclusion, besides confirming known effects of salinity on gills of euryhaline fish, molecular phenotyping reveals novel insight into proteome changes that underlie the remodeling of tilapia gill epithelium in response to environmental salinity change. A two-tiered label-free quantitative (LFQ) proteomics workflow was used to elucidate how salinity affects the molecular phenotype, i.e. proteome, of gills from a cichlid fish, the euryhaline tilapia (Oreochromis mossambicus). The workflow consists of initial global profiling of relative tryptic peptide abundances in treated versus control samples followed by targeted identification (by MS/MS) and quantitation (by chromatographic peak area integration) of validated peptides for each protein of interest. Fresh water acclimated tilapia were independently exposed in separate experiments to acute short-term (34 ppt) and gradual long-term (70 ppt, 90 ppt) salinity stress followed by molecular phenotyping of the gill proteome. The severity of salinity stress can be deduced with high technical reproducibility from the initial global label-free quantitative profiling step alone at both peptide and protein levels. However, an accurate regulation ratio can only be determined by targeted label-free quantitative profiling because not all peptides used for protein identification are also valid for quantitation. Of the three salinity challenges, gradual acclimation to 90 ppt has the most pronounced effect on gill molecular phenotype. Known salinity effects on tilapia gills, including an increase in the size and number of mitochondria-rich ionocytes, activities of specific ion transporters, and induction of specific molecular chaperones are reflected in the regulation of abundances of the corresponding proteins. Moreover, specific protein isoforms that are responsive to environmental salinity change are resolved and it is revealed that salinity effects on the mitochondrial proteome are nonuniform. Furthermore, protein NDRG1 has been identified as a novel key component of molecular phenotype restructuring during salinity-induced gill remodeling. In conclusion, besides confirming known effects of salinity on gills of euryhaline fish, molecular phenotyping reveals novel insight into proteome changes that underlie the remodeling of tilapia gill epithelium in response to environmental salinity change. Euryhaline fish are capable of living in fresh water (FW), 1The abbreviations used are:FWfresh waterACaccession numberACNacetonitrileBPCbase peak chromatogramBWbrackish waterCAcarbonic anhydraseEICextracted ion chromatogramERKextracellular signal-regulated kinaseFAformic acidGSK3glycogen synthase kinase 3HAV-type H+-ATPaseTICtotal ion chromatogramLFQlabel-free quantitationNDRG1N-myc downstream regulated gene 1NEDD4neural precursor cell expressed developmentally down-regulated protein 4NKANa+/K+-ATPaseNKCCNa+/K+/Cl− cotransporterPI3Kphosphoinositide 3-kinasePCAprincipal component analysispptparts per thousandRasRat sarcoma proteinSGK1serum and glucocorticoid inducible kinase 1SMADsmall body size/mothers against decapentaplegic homologSWseawater. 1The abbreviations used are:FWfresh waterACaccession numberACNacetonitrileBPCbase peak chromatogramBWbrackish waterCAcarbonic anhydraseEICextracted ion chromatogramERKextracellular signal-regulated kinaseFAformic acidGSK3glycogen synthase kinase 3HAV-type H+-ATPaseTICtotal ion chromatogramLFQlabel-free quantitationNDRG1N-myc downstream regulated gene 1NEDD4neural precursor cell expressed developmentally down-regulated protein 4NKANa+/K+-ATPaseNKCCNa+/K+/Cl− cotransporterPI3Kphosphoinositide 3-kinasePCAprincipal component analysispptparts per thousandRasRat sarcoma proteinSGK1serum and glucocorticoid inducible kinase 1SMADsmall body size/mothers against decapentaplegic homologSWseawater. brackish water (BW), seawater (SW), and hypersaline water (>SW). They adjust transepithelial ion transport across gill epithelium when challenged by an environmental salinity change (1Evans D.H. Teleost fish osmoregulation: what have we learned since August Krogh, Homer Smith, and Ancel Keys.Am. J. Physiol. 2008; 295: R704-R713Crossref PubMed Scopus (272) Google Scholar). Acclimation from hyposmotic (relative to plasma, e.g. FW) to hyperosmotic (relative to plasma, e.g. SW) environments is accompanied by extensive remodeling of gill epithelium, the most prominent feature of which is an increase in the number and size of salt-secretory, mitochondria-rich ionocytes (2Karnaky K.J. Structure and function of the chloride cell of Fundulus heteroclitus and other teleosts.Am. Zool. 1986; 26: 209-224Crossref Scopus (156) Google Scholar). In addition, molecular chaperones and distinct sets of transport proteins are activated when euryhaline fish are challenged by increasing environmental salinity (3Kültz D. Plasticity and stressor specificity of osmotic and heat shock responses of Gillichthys mirabilis gill cells.Am. J. Physiol.-Cell. 1996; 271: C1181-C1193Crossref PubMed Google Scholar, 4Dymowska A.K. Hwang P.P. Goss G.G. Structure and function of ionocytes in the freshwater fish gill.Resp. Physiol. Neurobiol. 2012; 184: 282-292Crossref PubMed Scopus (155) Google Scholar, 5Galvez F. Reid S.D. Hawkings G. Goss G.G. Isolation and characterization of mitochondria-rich cell types from the gill of freshwater rainbow trout.Am. J. Physiol.-Reg. Int. Comp. Physiol. 2002; 282: R658-R668Crossref PubMed Scopus (105) Google Scholar). A euryhaline fish species in which these physiological responses have been observed is the Mozambique tilapia, Oreochromis mossambicus (6Kültz D. Bastrop R. Jürss K. Siebers D. Mitochondria-rich (MR) Cells and the activities of the Na+/K+-ATPase and carbonic anhydrase in the gill and opercular epithelium of Oreochromis mossambicus adapted to various salinities.Comp. Biochem. Physiol. B. 1992; 102: 293-301Crossref Scopus (78) Google Scholar, 7Foskett J.K. Bern H.A. Machen T.E. Conner M. Chloride cells and the hormonal control of teleost fish osmoregulation.J. Exp. Biol. 1983; 106: 255-281Crossref PubMed Google Scholar, 8Kültz D. Jürss K. Jonas L. Cellular and epithelial adjustments to altered salinity in the gill and opercular epithelium of a cichlid fish (Oreochromis mossambicus).Cell Tissue Res. 1995; 279: 65-73Crossref Scopus (50) Google Scholar). Tilapia have evolved in Africa but have spread to subtropical and tropical freshwater and marine habitats throughout the world as a result of escaping from aquaculture farms and their high environmental adaptability. These cichlids tolerate salinities ranging from fresh water to almost 4× seawater (120 ppt) and they inhabit freshwater and hypersaline desert lakes as well as coastal marine and brackish habitats (9Stickney R.R. Tilapia tolerance of saline waters - a review.Prog. Fish Cult. 1986; 48: 161-167Crossref Google Scholar). This high salinity tolerance may have been selected for during tilapia evolution by frequent seasonal droughts and intermittent flooding events in their native African habitat containing salt-rich bedrock and soil (10Costa-Pierce B.A. Rapid evolution of an established feral tilapia (Oreochromis spp.): The need to incorporate invasion science into regulatory structures.Biol. Invasions. 2003; 5: 71-84Crossref Scopus (83) Google Scholar). Tilapia are highly abundant in the California Salton Sea, which is a large hypersaline desert lake with an average salinity of 50 ppt and seasonal salinity increases up to 100 ppt in some parts (11Sardella B.A. Brauner C.J. Cold temperature-induced osmoregulatory failure: The physiological basis for tilapia winter mortality in the Salton Sea?.Calif. Fish Game. 2007; 93: 200-213Google Scholar, 12Miles K.A. Ricca M.A. Meckstroth A. Spring S.E. Salton Sea Ecosystem Monitoring Project.U.S. Geol. Survey Open-File Rep. 2009; 1276: 1-150Google Scholar, 13Sardella B.A. Matey V. Cooper J. Gonzalez R.J. Brauner C.J. Physiological, biochemical and morphological indicators of osmoregulatory stress in 'California' Mozambique tilapia (Oreochromis mossambicus x O. urolepis hornorum) exposed to hypersaline water.J. Exp. Biol. 2004; 207: 1399-1413Crossref PubMed Scopus (106) Google Scholar). Thus, studies investigating the mechanisms that enable tilapia to cope with extreme and diverse osmotic stress are of great interest from an ecophysiological perspective and for understanding the basis of their high invasiveness in novel habitats. fresh water accession number acetonitrile base peak chromatogram brackish water carbonic anhydrase extracted ion chromatogram extracellular signal-regulated kinase formic acid glycogen synthase kinase 3 V-type H+-ATPase total ion chromatogram label-free quantitation N-myc downstream regulated gene 1 neural precursor cell expressed developmentally down-regulated protein 4 Na+/K+-ATPase Na+/K+/Cl− cotransporter phosphoinositide 3-kinase principal component analysis parts per thousand Rat sarcoma protein serum and glucocorticoid inducible kinase 1 small body size/mothers against decapentaplegic homolog seawater. fresh water accession number acetonitrile base peak chromatogram brackish water carbonic anhydrase extracted ion chromatogram extracellular signal-regulated kinase formic acid glycogen synthase kinase 3 V-type H+-ATPase total ion chromatogram label-free quantitation N-myc downstream regulated gene 1 neural precursor cell expressed developmentally down-regulated protein 4 Na+/K+-ATPase Na+/K+/Cl− cotransporter phosphoinositide 3-kinase principal component analysis parts per thousand Rat sarcoma protein serum and glucocorticoid inducible kinase 1 small body size/mothers against decapentaplegic homolog seawater. Moreover, because of their outstanding osmotolerance tilapia are excellent models for studying the mechanisms of body water and electrolyte homeostasis in vertebrates. O. mossambicus is a very close relative of (and readily hybridizes with) the Nile tilapia, Oreochromis nilotics, for which a complete reference proteome is available in major databases, including UniProtKB (14D'Amato M.E. Esterhuyse M.M. van der Waal B.C.W. Brink D. Volckaert F.A.M. Hybridization and phylogeography of the Mozambique tilapia Oreochromis mossambicus in southern Africa evidenced by mitochondrial and microsatellite DNA genotyping.Conserv. Genet. 2007; 8: 475-488Crossref Scopus (83) Google Scholar, 15Wang W.S. Hung S.W. Lin Y.H. Tu C.Y. Wong M.L. Chiou S.H. Shieh M.T. Purification and localization of nitric oxide synthases from hybrid tilapia (Nile tilapia x Mozambique tilapia).J. Aquat. Anim. Health. 2007; 19: 168-178Crossref PubMed Scopus (13) Google Scholar). Therefore, tilapia are well suited for proteomics studies directed at identifying, quantifying, and explaining molecular phenotypes (alterations in the proteome) induced by environmental stress. Because higher-order phenotypes (physiology, morphology, behavior) associated with salinity acclimation are well documented for tilapia, knowledge of the underlying molecular phenotypes will provide insight into the mechanisms that govern salinity acclimation of euryhaline fish (13Sardella B.A. Matey V. Cooper J. Gonzalez R.J. Brauner C.J. Physiological, biochemical and morphological indicators of osmoregulatory stress in 'California' Mozambique tilapia (Oreochromis mossambicus x O. urolepis hornorum) exposed to hypersaline water.J. Exp. Biol. 2004; 207: 1399-1413Crossref PubMed Scopus (106) Google Scholar, 16Grau E.G. Helms L.M. The tilapia prolactin cell: twenty-five years of investigation.Prog. Clin. Biol. Res. 1990; 342: 534-540PubMed Google Scholar, 17Cataldi E. Mandich A. Ozzimo A. Cataudella S. The interrelationships between stress and osmoregulation in a euryhaline fish, Oreochromis mossambicus.J. Appl. Ichthyol. 2005; 21: 229-231Crossref Scopus (17) Google Scholar). The main purpose of this study is to optimize and use a label-free quantitative proteomics (LFQ) workflow for molecular phenotyping of tilapia gill responses to salinity stress. The workflow consists of initial protein identification and global label-free quantitative (LFQ) profiling followed by subsequent targeted LFQ of particular proteins based on quantitatively diagnostic, validated peptide ions. High resolution and high retention-time reproducibility in nano-flow liquid chromatography in combination with fast, high mass accuracy and high resolution mass spectrometers have enabled large-scale LFQ of proteins (18Ong S.E. Mann M. Mass spectrometry-based proteomics turns quantitative.Nature Chem. Biol. 2005; 1: 252-262Crossref PubMed Scopus (1317) Google Scholar). Both relative and absolute LFQ of proteins are possible (19Bostanci N. Heywood W. Mills K. Parkar M. Nibali L. Donos N. Application of label-free absolute quantitative proteomics in human gingival crevicular fluid by LC/MS E (gingival exudatome).J. Proteome Res. 2010; 9: 2191-2199Crossref PubMed Scopus (105) Google Scholar, 20Cutillas P.R. Vanhaesebroeck B. Quantitative profile of five murine core proteomes using label-free functional proteomics.Mol. Cell. Proteomics. 2007; 6: 1560-1573Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) and protein quantities can be inferred from either spectral counts or ion currents and chromatographic peak intensity (21Matzke M.M. Brown J.N. Gritsenko M.A. Metz T.O. Pounds J.G. Rodland K.D. Shukla A.K. Smith R.D. Waters K.M. McDermott J.E. Webb-Robertson B.J. A comparative analysis of computational approaches to relative protein quantification using peptide peak intensities in label-free LC-MS proteomics experiments.Proteomics. 2013; 13: 493-503Crossref PubMed Scopus (61) Google Scholar, 22Mallick P. Kuster B. Proteomics: a pragmatic perspective.Nat. Biotechnol. 2010; 28: 695-709Crossref PubMed Scopus (319) Google Scholar). Spectral counting procedures have been used to roughly approximate relative protein quantities in different samples (23Ishihama Y. Oda Y. Tabata T. Sato T. Nagasu T. Rappsilber J. Mann M. Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein.Mol. Cell. Proteomics. 2005; 4: 1265-1272Abstract Full Text Full Text PDF PubMed Scopus (1635) Google Scholar). In the present study, the other approach for LFQ, quantitation of ion current intensity, is used for relative quantitation of protein abundances in gill tissue from salinity stressed fish compared with FW handling controls. The quantitative precision of carefully optimized ion current intensity-based LFQ approaches is comparable to that of isotopic label-based quantitation (24Cutillas P.R. Geering B. Waterfield M.D. Vanhaesebroeck B. Quantification of gel-separated proteins and their phosphorylation sites by LC-MS using unlabeled internal standards: analysis of phosphoprotein dynamics in a B cell lymphoma cell line.Mol. Cell. Proteomics. 2005; 4: 1038-1051Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 25Old W.M. Meyer-Arendt K. Aveline-Wolf L. Pierce K.G. Mendoza A. Sevinsky J.R. Resing K.A. Ahn N.G. Comparison of label-free methods for quantifying human proteins by shotgun proteomics.Mol. Cell. Proteomics. 2005; 4: 1487-1502Abstract Full Text Full Text PDF PubMed Scopus (1017) Google Scholar). Ion current intensity can be measured as peak height (maximum ion current) or peak area (integral of extracted ion chromatogram) (20Cutillas P.R. Vanhaesebroeck B. Quantitative profile of five murine core proteomes using label-free functional proteomics.Mol. Cell. Proteomics. 2007; 6: 1560-1573Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 21Matzke M.M. Brown J.N. Gritsenko M.A. Metz T.O. Pounds J.G. Rodland K.D. Shukla A.K. Smith R.D. Waters K.M. McDermott J.E. Webb-Robertson B.J. A comparative analysis of computational approaches to relative protein quantification using peptide peak intensities in label-free LC-MS proteomics experiments.Proteomics. 2013; 13: 493-503Crossref PubMed Scopus (61) Google Scholar, 22Mallick P. Kuster B. Proteomics: a pragmatic perspective.Nat. Biotechnol. 2010; 28: 695-709Crossref PubMed Scopus (319) Google Scholar). Because peak area provides a more accurate measure of peptide (and correspondingly protein) quantity this approach is used in the present study (21Matzke M.M. Brown J.N. Gritsenko M.A. Metz T.O. Pounds J.G. Rodland K.D. Shukla A.K. Smith R.D. Waters K.M. McDermott J.E. Webb-Robertson B.J. A comparative analysis of computational approaches to relative protein quantification using peptide peak intensities in label-free LC-MS proteomics experiments.Proteomics. 2013; 13: 493-503Crossref PubMed Scopus (61) Google Scholar, 22Mallick P. Kuster B. Proteomics: a pragmatic perspective.Nat. Biotechnol. 2010; 28: 695-709Crossref PubMed Scopus (319) Google Scholar). The present study applies this LFQ workflow to identify the specific isoforms of (a) proteins involved in transepithelial ion transport and (b) molecular chaperones that are regulated by environmental salinity in tilapia gills. Such information is very difficult and often impossible to obtain with antibody-based approaches because isoform-specific antibodies for fish proteins are rare and none are available for the tilapia proteins of interest. Therefore, most quantitative analyses of fish proteins by Western blot use antibodies made against a different species (or even a mammalian or other more distantly evolutionarily related homolog) that are not suitable to distinguish individual isoforms (e.g. 3Kültz D. Plasticity and stressor specificity of osmotic and heat shock responses of Gillichthys mirabilis gill cells.Am. J. Physiol.-Cell. 1996; 271: C1181-C1193Crossref PubMed Google Scholar, 26Hofmann G.E. Buckley B.A. Airaksinen S. Keen J.E. Somero G.N. Heat shock protein expression is absent in the Antarctic fish Trematomus bernacchii (family Nototheniidae).J. Exp. Biol. 2000; 203: 2331-2339Crossref PubMed Google Scholar, 27Ojima N. Mekuchi M. Ineno T. Tamaki K. Kera A. Kinoshita S. Asakawa S. Watabe S. Differential expression of heat-shock proteins in F2 offspring from F1 hybrids produced between thermally selected and normal rainbow trout strains.Fisheries Sci. 2012; 78: 1051-1057Crossref Scopus (8) Google Scholar). The present study also investigates whether salinity-induced changes in ionocyte number and size are reflected in abundances of mitochondrial proteins, whether there is disparity in how different mitochondrial proteins are regulated in response to salinity stress, and which mitochondrial proteins are most affected by salinity stress. In addition, the initial global profiling step of the LFQ proteomics workflow described and the deposition of corresponding identification and quantitation data in the public PRIDE repository (28Csordas A. Ovelleiro D. Wang R. Foster J.M. Rios D. Vizcaino J.A. Hermjakob H. PRIDE: quality control in a proteomics data repository.Database. 2012; 2012: bas004Crossref PubMed Scopus (35) Google Scholar, 29Vizcaino J.A. Cote R.G. Csordas A. Dianes J.A. Fabregat A. Foster J.M. Griss J. Alpi E. Birim M. Contell J. O'Kelly G. Schoenegger A. Ovelleiro D. Perez-Riverol Y. Reisinger F. Rios D. Wang R. Hermjakob H. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013.Nucleic Acids Res. 2013; 41: D1063-D1069Crossref PubMed Scopus (1594) Google Scholar) provides quantitative information on many proteins for which no prior information about effects of salinity on their abundance is available. All animal procedures were approved by the UC Davis IACUC under protocol numbers 13468 and 15013. Mozambique tilapia (Oreochromis mossambicus) were raised from laboratory brood stock to between 5 and 6 months of age in dechlorinated Davis tap water for all experiments. Davis tap water chemistry has been reported previously (30Fiol D.F. Sanmarti E. Lim A.H. Kültz D. A novel GRAIL E3 ubiquitin ligase promotes environmental salinity tolerance in euryhaline tilapia.Biochim. Biophys. Acta. 2011; 1810: 439-445Crossref PubMed Scopus (13) Google Scholar), having a salinity < 0.5 ppt (fresh water, FW). Three different types of salinity acclimation were conducted in which fish were exposed to either acute salinity increase from FW to 34 ppt (in two 17 ppt/day steps) or gradual salinity increase from FW to 70 ppt and from FW to 90 ppt (in 7 ppt/day steps) (Fig. 1A). All acclimation experiments were performed at 26 ± 1 °C in 30 gallon closed circulation tanks equipped with appropriate aeration, filtration, and heating devices. Daily changes of 10% of the tank volume of water with a stock solution of sea salt (Instant Ocean) were performed to increase salinity by appropriate increments. Handling controls were run in parallel in each experiment. These controls consisted of fish from the same clutch that were kept in identical tanks and exposed to the same daily water changes except that FW was used instead of a concentrated sea salt solution. Fish were fed ad libitum and continued to grow well during salinity stress except at very high salinities (90 ppt exposure) when body mass (but not total length) was significantly less than that of the corresponding FW handling controls (Figs. 1B, 1C). A protocol that minimizes sample preparation steps while efficiently and reproducibly extracting membrane-, cytosolic-, and organelle-specific proteins was developed and optimized to maximize quantitative accuracy for comparing protein amounts in multiple samples. Immediately after dissection tissue samples were snap-frozen in liquid nitrogen and transferred to −80 °C. They were crushed to a fine powder by grinding the tissue using a mortar and pestle under liquid nitrogen. The powder was transferred to a low retention Eppendorf tube (LR-MCF) and an ice-cold solution of 10% trichloroacetic acid (TCA)/90% Acetone/0.2% dithiothreitol (DTT) was added (6× the volume of tissue weight). Samples were incubated in this solution while rotating overnight. Samples were then centrifuged at 20,000 × g for 5 min (4 °C) and the precipitated protein pellet washed twice in acetone/0.2% DTT. The protein pellet was dissolved in UT buffer (7 m urea/2 M thiourea/0.2% DTT, 6x the volume of the original tissue weight). After centrifugation at 18,000 × g (5 min) the supernatant was transferred to a clean LR-MCF tube and stored frozen at −80 °C. Before freezing the sample, triplicate 2 μl aliquots were used to determine protein concentration using a 660 nm protein assay that is compatible with 5× diluted UT buffer (Thermo-Pierce, cat. 22660). Protein extracts were thawed and appropriate amounts of LCMS grade water and 10× triethanolamine (TEA) buffer (pH 8.0, final concentration 100 mm) added to dilute samples of 150 μg/100 μl total protein concentration in 0.6 ml LR-MCF tubes. Samples were reduced by adding 16 mm DTT and incubation at 55 °C for 30 min, and alkylated by adding 16 mm iodoacetic acid (IAA) followed by 30 min incubation at room temperature in the dark. Immobilized trypsin (Princeton Separations cat. Nr. EN-251) was added at a 1:25 ratio relative to total protein and the samples incubated in a rotator at 35 °C for exactly 16h. Trypsin beads were removed by centrifugation for 2 min at 14,000 × g and 50% of the total initial volume of supernatant was transferred to a clean 0.6 ml LR-MCF tube. Samples were then dried by speedvac (Thermo-Savant, ISS-110). This step was stopped immediately when urea precipitate started to form to ensure optimal resuspension of peptides, which was done in 1 ml of LCMS grade water containing 0.1% formic acid (FA). Pipetting the solution slowly 50× up and down using a pipetman set at 0.8 ml over a period of 5 min ensured that peptides and urea redissolved. Peptide solutions were then transferred to maximum recovery glass vials (Waters 600000669CV) and stored frozen at −80 °C. Peptides were injected in 2 μl volume corresponding to 330 ng total peptide amount using a nanoAcquity sample manager (Waters, Milford, MA) and separated after 3 min sample trapping (Symmetry, Waters 186003514) on a 1.7 μm particle size BEH C18 column (250 mm × 75 μm, Waters 186003545) by reversed phase chromatography using a nanoAcquity binary solvent manager (Waters). A 50 min linear gradient ranging from 3% to 35% acetonitrile (ACN) was used. The aqueous solvent contained 0.1% FA, which was omitted from the organic phase to prevent formation of brown ACN aggregates that would otherwise precipitate at low pH at the pico-emitter tip. A dual pico-emitter tip (New Objective FS360–20-10-d-20) nano-spray setup was custom-fitted at the nano-ESI source of a micrOTOF-QII mass spectrometer (Bruker Daltonics, for details see PRIDE AC 28628) to deliver analyte via two independent solvent lines. Line 1 was used for the sample gradient and controlled by the binary solvent manager of the nanoAcquity UPLC. Line 2 was used to deliver ESI-L low concentration tuning mix (Agilent G1969–85000) independent of line 1 during the 20 min sample injection delay period to allow for internal mass calibration of each sample. Internal calibration performed in this manner yielded better and more consistent mass accuracy across multiple samples than lock-mass calibration. Batch-processing of samples was controlled with Hystar 3.2 software (Bruker Daltonics). All peaklists were generated with Mascot Daemon and Mascot Distiller (versions 2.2.2 and 2.4.3, respectively, Matrixscience. Ltd.) using default parameters. For protein identification Mascot 2.2.7 (Matrixscience, Ltd.) (31Perkins D.N. Pappin D.J. Creasy D.M. Cottrell J.S. Probability-based protein identification by searching sequence databases using mass spectrometry data.Electrophoresis. 1999; 20: 3551-3567Crossref PubMed Scopus (6763) Google Scholar) and Phenyx 2.6. (Geneva Bioinformatics, SA) (32Masselot A. Binz P.A. Cambria L. Appel R.D. Phenyx: Combining high-throughput and pertinence in protein identification.Mol. Cell. Proteomics. 2004; 3: S257PubMed Google Scholar) search engines were used and search results combined in Proteinscape 3.1 (Bruker Daltonics). The following parameters were used: enzyme specificity = trypsin, missed cleavages permitted = 1, fixed modification = Cys carbamidomethylation, variable modifications = Met oxidation and Pro hydroxylation, precursor ion mass tolerance = 20 ppm, fragment ion mass tolerance = 0.1 Da. A threshold score of 5% probability that a protein identification is incorrect was used for accepting individual MS/MS spectra. A database containing 28,020 protein sequences, including the complete predicted Oreochromis niloticus proteome and all available Oreochromis mossambicus sequences, was downloaded from http://www.uniprot.org/on July 28, 2012. Automatic annotation of all Uniprot ACs in this database was done with BLAST2GO (33Götz S. Garcia-Gómez J.M. Terol J. Williams T.D. Nagaraj S.H. Nueda M.J. Robles M. Talón M. Dopazo J. Conesa A. High-throughput functional annotation and data mining with the Blast2GO suite.Nucleic Acids Res. 2008; 36: 3420-3435Crossref PubMed Scopus (2886) Google Scholar) against the complete curated SwissProt database on July 28, 2012. In addition, annotations for all proteins identified in this study were manually confirmed by individual BLAST searches. An expanded version of the Oreochromis spec. protein database, which contains a randomly scrambled decoy sequence for each entry, was generated using PEAKS 6 (Bioinformatics Solutions, Inc.) (34Zhang J. Xin L. Shan B.Z. Chen W.W. Xie M.J. Yuen D. Zhang W.M. Zhang Z.F. Lajoie G.A. Ma B. PEAKS DB: De Novo Sequencing Assisted Database Search for Sensitive and Accurate Peptide Identification.Mol. Cell. Proteomics. 2012; : 11Google Scholar). This expanded decoy database (containing 56,040 total sequences) was used for all protein identification searches to allow for consistent assessment of the protein ID false discovery rate. Redundancy in assigning peptides to protein identifications and ambiguity in protein identifications were eliminated by ProteinExtractor (Proteinscape 3.1). All data and metadata were exported from Proteinscape 3.1 to PRIDE xml using PRIDE
Referência(s)