TMT-Based Proteomic Analysis of Scophthalmus maximus Hearts Under High Temperature Stress

Water temperature is one of the most important ecological environmental factors affecting fish survival and growth. When the water temperature is higher than the optimal temperature for fish survival, fish homeostasis may be disrupted. The heart plays a crucial role in maintaining vertebrate homeostasis. In this study, TMT and LC-MS/MS were performed to analyze differentially expressed protein (DEP) profiles in the hearts of turbot under high-temperature stress to explore the key proteins and metabolic pathways related to the high-temperature tolerance traits of turbot. Ninety turbot (mean weight 23.01±3.63 g, mean length 10.34±0.76 cm) were used in experiments. Three temperature points for heat stress experiments were designed: control=(14.0±0.5)℃, HT1=(20.0±0.5)℃, and HT2=(28.0±0.5)℃. Three parallel groups were established for each temperature group, with 10 turbots placed in each group. Fold Change≥1.2 or < 0.83, P < 0.05 was used as a criterion to screen for DEPs in turbot. Through mass spectrometry analysis of heart tissues from turbot, 85, 731 spectra were identified, with 35, 434 peptides, of which 31, 704 were unique, and 4, 765 proteins were identified. The HT1/control group in the heart of turbot had a total of 87 differentially expressed proteins, including 77 upregulated and 10 down-regulated proteins; the HT2/control group in the heart of turbot had 171 DEPs, including 149 upregulated and 22 down-regulated proteins; the HT2/HT1 group in the heart of turbot had a total of 188 DEPs, including 130 upregulated and 58 downregulated proteins. Through bioinformatics analysis, the DEPs between the 28℃ group and the 14℃ group, as well as the 28℃ group and the 20℃ group, were mainly enriched in KEGG pathways related to fatty acid metabolism, inflammation, and immune defense. Multiple proteins in the heat shock protein family (HSP90B, HSP70, etc.) were significantly upregulated in the 28℃ group compared to the 14℃ group. The protein interaction network revealed that HSP90B exhibited the highest correlation. A new discovery is that the expression levels of fibrinogen-related proteins (FGA, FGB, and FGG) increased, and their role in the thermal response requires further research. These results indicate that extremely high temperatures may cause fatty acid metabolic disorders in turbot, leading to inflammatory reactions. The upregulation of heat shock protein family proteins may have a positive effect on alleviating stress symptoms under heat stress. This study identified DEPs, such as HSP90B, HSPA5, and HYOU1, which were significantly upregulated under heat stress, as candidate proteins for high-temperature tolerance breeding in turbot and provided a theoretical basis for further elucidating the molecular mechanism of heat stress in turbot.