Supplementary figures:
Supplementary figure 1: Salinity exposure protocols used to determine collect samples at relevant salinity-level/duration points before LER. To determine CSMAX, salinity was raised at five different rates of change per day (32, 24, 12, 8, and 6g/kg/day) until LER. To determine CSMAX over time at hypersalinity, salinity was increased at a rate of 6g/kg/day to three target salinity levels (85, 95, and 105g/kg) followed by continuous exposure at target salinity until LER. To assess physiological and molecular function at hypersalinity before LER, salinity was raised to four target levels (21g/kg, 55g/kg, 85g/kg, and 105g/kg) over14 days and samples collected following 24 hours at the final salinity. To assess the long-term impacts of hypersalinity below the critical salinity threshold, fish were acclimated from FW to 75g/kg at 6g/kg/day over 14 days followed by maintenance at the final salinity for 10 weeks for a 12 week acclimation in total. Samples used for proteomic analysis, chosen as representative of points above and below the critical salinity threshold, are circled in red.
Supplementary figure 2 : Properties of a O. mossambicusgill epithelium DIA assay library relative to the corresponding raw spectral library. The initial spectral library (SL) represents over 16,000 proteins, 139,000 precursors, and 864,000 transitions. Ten QC filters were applied to create the DIA assay library containing 3024 proteins (A), 13,847 peptides (B) and precursors (C), and 68,586 transitions (D). Most proteins are represented by at least 2 diagnostic peptides. The remainder (825) was identified by at least 2 unique peptides but only 1 remains after applying all DIA QC filters. The initial bar labeled SL depicts data for the raw spectral library and final bar (step 10) depicts data for the DIA assay library. Library filtration steps one to six are explained in the text. (E), Frequency distributions of fragment ion types represented in the final DIA assay library. b+ ions are fragments which extend from the N-terminus of the peptide and y+ ions extend from the C-terminus i.e. a 3 fragment ion denominator member contains the first three peptides for a b+ ion and the last three for a y+ ion. (F), Frequency distribution for the number of peptides per protein in the DIA assay library. The data were generated with Skyline 20.0 (MacCoss Lab., University of Washington).
Supplementary figure 3 : Quality control parameters summarized for all samples in five treatments analyzed in this study. A) The mean mass error (ppm) for all transitions present in the final assay library in the 14-day 85g/kg treatment. Other treatments shown are 14-day 105g/kg (E), extended 85g/kg (I), extended 105g/kg (M), and 12-week 75g/kg (Q). B) Retention time for the 14-day 85g/kg treatment. reproducibility for all transitions in all samples analyzed in this study was very high (r2 = 1.0). Other treatments shown are 14-day 105g/kg (F), extended 85g/kg (J), extended 105g/kg (N), and 12-week 75g/kg (R). C) Fold change (FC) and coefficient of variation (CV) depending on number of biological replicates at a statistical power of 0.8 and false discovery rate (FDR) of 1% in the 14-day 85g/kg treatment. Other treatments shown are 14-day 105g/kg (G), extended 85g/kg (K), extended 105g/kg (O), and 12-week 75g/kg (S). D) mProphet Q values for all transitions in all samples in the 14-day 85g/kg treatment. Other treatments shown are 14-day 105g/kg (H), extended 85g/kg (L), extended 105g/kg (P), and 12-week 75g/kg (T). Figures were generated with Skyline 20.0 (including MSstats and mProphet) software (MacCoss Lab., University of Washington).
Supplementary figure 4: Alignment and distance tree of full uncharacterized protein sequence and one repeat of the sequence. A) Alignment of the full sequence of the uncharacterized protein LOC100699110 - X1 with all named proteins in the top 100 blastp results based on E-value using the non-redundant protein sequence database of the NCBI and no specified organism. B) Alignment of the 143 amino acid (AA) repeat segment using the same parameters as in A. C) Distance tree of results from A based on BLAST computed pairwise alignment with evolutionary distance modeled as the expected fraction of AA substitutions per site based on the fraction of mismatched AAs in the region following [64]. D) Distance tree of results for 143 AA repeat results. E) Beginning section of the amino acid sequence showing peptide coverage from the DIA assay library. Repeat AA segment is highlighted.