Two commercial diet formulations (labelled as Diet A and Diet B) were fed to barramundi over 12 weeks as part of a feeding trial to evaluate the impact of diet on protein expression in brain, liver and intestine. Fish were fed twice daily, water quality including oxygen, salt, ammonium and pH were checked daily, and fish growth and performance measurements were performed daily. Proteins were extracted in modified RIPA buffer, and proteomic analysis was performed using DIA on a Q Exactive HFX, with data analysis performed using DIA-NN and Fragpipe Analyst.
In total, 3,939 proteins were quantified in the brain, 3,792 in liver, and 5,072 in the intestine. A total of 206 upregulated and 519 downregulated proteins were identified in the brain, 311 upregulated and 212 downregulated proteins in the liver, and 611 upregulated and 404 downregulated proteins in the intestine.
Gene enrichment analysis of differentially expressed proteins in the brain revealed significant alterations in metabolic pathways, particularly those related to energy production and oxidative metabolism. Notably, the apelin signalling pathway, which regulates feed intake, was upregulated. In the liver, pathways related to amino acid biosynthesis, general metabolism, and tryptophan metabolism were downregulated. Additionally, KEGG pathway analysis indicated upregulation of necroptosis and ferroptosis — both of which are forms of regulated cell death. Ferritin was identified among the proteins involved in ferroptosis pathway.
ICP-MS analysis of liver tissue further confirmed a significantly higher iron (Fe) concentration in fish fed on diet B (0.126 ± 0.057 mg/g) compared to diet A (0.072 ± 0.021 mg/g), aligning with the proteomic findings. These results highlight that diet B, associated with lower feed intake, contains compounds and nutrients that negatively impact the health and growth performance of barramundi compared to diet A.