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.