A data-independent acquisition assay library was generated for the liver of threespine sticklebacks to evaluate alterations in protein abundance and functional enrichment of molecular pathways following either a chronic warm (25°C) or cold (7°C) three-week temperature challenge in two estuarine populations. The assay library was created from a data-dependent acquisition (DDA) based raw spectral library that was filtered to remove low quality or ambiguous peptides. Functional enrichment analyses using STRING aided in identifying larger networks and domains that were significantly enriched in the different groups. The Big Lagoon population (BL) had a stronger response to both temperatures than the Klamath River population (KL). At 7°C, BL showed alterations in protein homeostasis that likely fueled a higher demand for energy, but both populations appeared to successfully acclimate to this temperature. The warm temperature acclimation saw major increases in proteins involved in chromatin structure, including a variety of histones, and transcription, while there were decreases in proteins related to translation and fatty acid metabolism. Functional enrichment analyses uncovered differences in metabolism and highlighted altered molecular pathways that only had a few representative proteins reach significance from the individual quantification analyses. This data set represents the unique proteomic signatures of two populations of threespine sticklebacks and helps elucidate general themes as well as highlight population differences in acclimating to chronic temperature stress. The synchronous expression patterns of numerous proteins gives better context and specificity to a particular stressor and helps identify the strategies and mechanisms utilized to overcome changes in the environment.

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 gradually acclimated (2°C increase or decrease per day) to either 25°C or 7°C and held at these temperatures for three weeks. Handling controls were kept at 15°C for the duration of the experiment. There were ten replicates per treatment.

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.