A data-independent acquisition assay library was used to examine proteome wide changes in liver of two populations of threespine stickleback (Gasterosteus aculeatus) after acute thermal stress. Experimental fish were held at 28°C for two hours and allowed to recover back at the acclimation temperature of 15°C for either six or 24 hours, while controls remained at 15°C. The Big Lagoon population (BL) had a large response at six hours after acute heat stress but had mostly regained protein homeostasis by 24 hours after the heat stress. The Klamath population showed very little response to acute thermal stress at either time point, highlighting major differences between the two populations in their response to acute heat stress. Functional enrichment analyses using STRING aided in identifying larger networks and domains that were significantly enriched in the different groups beyond the individual significantly different proteins, and KEGG pathway analysis was conducted on the large number of significantly different proteins for the BL28-6hr vs BL15-6hr comparison. DnaJ/Hsp40 B1b was the one protein that was significantly higher in abundance for both populations at both time points, suggesting a key role for this protein in regulating and orchestrating the response to acute temperature stress for this species. At six hours after acute heat stress, the main themes from the significant protein changes in the Big Lagoon population were the regulation of multiple levels of gene expression, reduction of reactive oxygen species (ROS) production, and structural changes.

First generation (F1) sticklebacks from Big Lagoon (Trinidad, CA) and Klamath River (Klamath, CA) were pre-acclimated for a minimum of three weeks at 15°C and 9 ppt prior to experimentation. Fish were exposed to 28°C for two hours, while controls were kept at 15°C for the duration of the experiment.

Replicates represent biological replicates, i.e. liver 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 150 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.