Physiological Genomics Group, Department of Animal Sciences, University of California Davis, One Shields Avenue, Davis, California 95616.
Mol Cell Proteomics. 2013 Dec;12(12):3962-75. doi: 10.1074/mcp.M113.029827. Epub 2013 Sep 24.
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.
采用双层非标记定量（LFQ）蛋白质组学工作流程来阐明盐度如何影响丽鱼（罗非鱼）鳃的分子表型，即蛋白质组。该工作流程包括对处理和对照样品中的相对胰蛋白酶肽丰度进行初始全局分析，然后对每个感兴趣的蛋白质进行靶向鉴定（通过 MS/MS）和定量（通过色谱峰面积积分）。淡水驯化的罗非鱼分别在单独的实验中独立暴露于急性短期（34 ppt）和逐渐长期（70 ppt、90 ppt）盐度胁迫下，然后对鳃蛋白质组进行分子表型分析。可以从初始的全局非标记定量分析步骤中单独推断出盐度胁迫的严重程度，在肽和蛋白质水平上都具有很高的技术重现性。然而，只有通过靶向非标记定量分析才能确定准确的调节比，因为并非所有用于蛋白质鉴定的肽也可用于定量。在三种盐度挑战中，逐渐适应 90 ppt 对鳃分子表型的影响最为显著。众所周知，盐度对罗非鱼鳃的影响，包括增加富含线粒体的离子细胞的大小和数量、特定离子转运体的活性以及诱导特定分子伴侣，都反映在相应蛋白质丰度的调节上。此外，还解析了对环境盐度变化有反应的特定蛋白质同工型，并揭示了盐度对线粒体蛋白质组的影响是不均匀的。此外，还鉴定出 NDRG1 蛋白是盐度诱导鳃重塑过程中分子表型重构的新关键组成部分。总之，除了证实盐度对广盐性鱼类鳃的已知影响外，分子表型还揭示了蛋白质组变化的新见解，这些变化是罗非鱼鳃上皮对环境盐度变化进行重塑的基础。
