Quantitative Molecular Phenotyping of Gill Remodeling in a Cichlid Fish Responding to Salinity Stress

Abstract

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. Molecular & Cellular Proteomics 12: 10.1074/ mcp.M113.029827, 3962-3975, 2013.Euryhaline fish are capable of living in fresh water (FW), 1 brackish water (BW), seawater (SW), and hypersaline water (ϾSW). They adjust transepithelial ion transport across gill epithelium when challenged by an environmental salinity change (1). 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 (2). In addition, molecular chaperones and distinct sets of transport proteins are activated when euryhaline fish are challenged by increasing environmental salinity (3)(4)(5). A euryhaline fish species in which these physiological responses have been observed is the Mozambique tilapia, Oreochromis mossambicus (6 -8). Tilapi...

Derivation and Osmotolerance Characterization of Three Immortalized Tilapia (Oreochromis mossambicus) Cell Lines

“…Peptides were injected in 2 µl volume corresponding to 330 ng total peptide amount using a nanoAcquity sample manager (Waters) 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 130 min linear solvent gradient ranging from 3% to 35% acetonitrile (ACN) was employed, internal calibration performed, and mass spectra acquired as previously described [37] except that an ImpactHD mass spectrometer (Bruker Daltonics) was used. Raw data were converted into mzXML format using DataAnalysis 4.2 (Bruker Daltonics) and PEAKS suite 7 (BSI, Inc.), Mascot 2.2.7 (Matrixscience, Ltd.), and X!Tandem Cyclone (The GPM) were used for protein identification using the following parameters: enzyme specificity  =  trypsin, max.…”

Salinity-induced activation of the myo-inositol biosynthesis pathway in tilapia gill epithelium

“…Tissue homogenization, protein extraction and in-solution digestion procedures were extensively optimized to yield quantitatively reproducible LC-MS/MS data and minimize protein modification during processing (Kültz et al, 2013). Each sample was homogenized under liquid nitrogen in a mortar and pestle.…”

Contrasting seasonal and aseasonal environments across stages of the annual cycle in the rufous‐collared sparrow, Zonotrichia capensis: Differences in endocrine function, proteome and body condition

“…Tryptic peptides were separated by nano‐ultra‐high‐performance liquid chromatography (UPLC) (Waters) using a 5–35% acetonitrile gradient over a 2‐hr retention time period and injected online into an Impact‐HD UHR‐qTOF mass spectrometer (Bruker Daltonics). Following electrospray ionization, peptides were identified using data‐independent acquisition and tandem mass spectrometry as previously described (Kültz, Li, Gardell, & Sacchi, ). Label‐free quantification was performed using peaksq 8.0 for an MS1 Top3 approach and scaffold 4.8 for a spectral counting approach as previously described (Kültz et al., , ).…”

“…Following electrospray ionization, peptides were identified using data‐independent acquisition and tandem mass spectrometry as previously described (Kültz, Li, Gardell, & Sacchi, ). Label‐free quantification was performed using peaksq 8.0 for an MS1 Top3 approach and scaffold 4.8 for a spectral counting approach as previously described (Kültz et al., , ). More details on methodology and parameters used for quantitative proteomics are provided in Supporting Information.…”