Proteomics of osmoregulatory versus general stress responses in threespine stickleback gills
Integ. and Comp. Biol.
- Organism: Gasterosteus aculeatus
- Instrument: impact II
Salinity, Fish, Osmoregulation, Stress, Gill, DIA
- Lab head:
The effect of environmental salinity on the gill proteome was isolated in controlled mesocosm experiments. Salinity-dependent changes in the gill proteome were analyzed by LCMSMS data-independent acquisition (DIA) and Skyline. For this purpose, relative abundances of 1691 proteins representing the molecular phenotype of stickleback gills were quantified using previously developed MSMS spectral and assay libraries in combination with data-independent acquisition (DIA) quantitative proteomics (Li et al. 2018). Osmoregulatory responses were distinguished from general stress responses based on the direction of protein abundance changes. If the abundance of a protein was consistently regulated in opposite directions by hyper- versus hypo-osmotic salinity stress in all six mesocosm experiments then it was considered an osmoregulatory protein. If protein abundance was always increased in all six mesocosm experiments independent of whether salinity was increased or decreased then it was considered a stress response protein. KEGG pathway analysis revealed that the inositol phosphate metabolism, salivary secretion, several endocrine and extracellular signaling, valine, leucine and isoleucine degradation, citrate cycle, and oxidative phosphorylation pathways contain most osmoregulatory gill proteins whose abundance is directly proportional to environmental salinity. Most proteins that were inversely correlated with salinity map to pathways that represent proteostasis, immunity and intracellular signaling processes. General stress response proteins represent fatty and amino acid degradation, purine metabolism, focal adhesion, mRNA surveillance, phagosome, endocytosis, and several intracellular signaling pathways. Some proteins in these general stress response pathways are consistently down-regulated instead of being up-regulated during any type of salinity stress indicating that these KEGG pathways are not uniformly induced or repressed but that their function is being altered more intricately.
Six independent mesocosm experiments were performed with fish collected in different years from Bodega Harbor, California (marine G. aculeatus trachurus) and Lake Solano, California (freshwater G. aculeatus leiurus). In these experiments fish were gradually acclimated (3 g/kg salinity change per day) to 1, 34, and 67 g/kg salinity. Handling controls kept at habitat salinity (34 g/kg for Bodega Harbor fish and 1 g/ kg for Lake Solano fish) were always included in parallel to all salinity treatments. Marine fish were exposed to hypo-osmotic salinity stress (34 --> 1 g/kg) in two experiments and to hyper-osmotic salinity stress (34 --> 67 g/kg) in another two experiments. Freshwater fish were only exposed to hyperosmotic salinity stress in two additional experiments (1 --> 34 g/kg and 1 --> 67 g/kg). The number of replicates per treatment or control group varied between six and twelve depending on the experiment but was always the same for treatments and control pairs within an experiment.
Replicates represent biological replicates, i.e. gill tissue isolated from different fish. Samples were processed by protein extraction, in-solution digestion with immobilized trypsin, and buffer exchange as previously described (Kültz et al., 2013, Mol Cell Proteomics 12, 3962-3975). An amount of 200 ng total peptide mix dissolved in 0.1% formic acid was injected into a nanoAcquity sample manager (Waters) and separated by reversed phase chromatography (300 nL/min flow rate) using a nanoAcquity binary solvent manager (Waters) prior to online nanoESI.
Created on 2/19/20, 9:08 PM