Proteome regulation during salinity stress
Proteomic analysis was performed for seven treatments using the same DIA assay library. The mass error threshold was <20 ppm for all transitions in all samples and <10 ppm for the great majority, and retention time reproducibility of the data was very high as well (Supplementary fig. 2). A fold-change (FC) threshold of 2.0 was enforced for all treatments in considering statistical significance based on the coefficient of variation calculated with MSstats (Choi et al., 2014), providing at least 0.8 statistical power for p-values >0.05. Furthermore, the majority of transition peaks for all samples in this dataset had mProphet (Reiter et al., 2011) peak scores of q<0.01, the peak quality threshold for inclusion in MSstats quantitative DIA data analysis.
Acclimation to 85g/kg resulted in 234 significantly upregulated and 171 significantly downregulated out of 2971 total proteins quantified, while acclimation to 105g/kg yielded 348 significantly upregulated and 255 significantly downregulated out of 2972 total proteins quantified (Figure 3A, Supplementary table 1). Extended exposure resulted in 501 significantly upregulated and 481 significantly downregulated out of 3015 proteins quantified at MP in 85g/kg, 486 significantly upregulated and 473 significantly downregulated out of 3015 proteins quantified at MP in 105g/kg, and 311 significantly upregulated and 149 significantly downregulated out of 3011 proteins quantified after ten weeks at 75g/kg.
The ten most highly upregulated and downregulated significant proteins based on FC were determined for each treatment and compared between treatments (Figure 3B). The most highly upregulated protein in all extended constant salinity treatments and the second most highly upregulated in the 14-day acclimations was inositol monophosphatase 1 isoform X1 (IMPase1-X1), which had a maximum upregulation in the extended 85g/kg salinity treatment of 438 FC and an average FC increase of 225 across all five treatments. Solute carrier family 12 member 2 isoform X1 (SLC12a2-X1) was the highest upregulated protein in both 14-day acclimations and the second most highly upregulated protein in the extended treatments, with an average of 90 times greater across all treatments. The most highly downregulated protein in four of the five treatments was an uncharacterized protein, LOC100699110 isoform X1, which was 1137 times lower in the extended 105g/kg exposure and 513 times lower on average across all treatments.
There was a large degree of overlap in significantly regulated proteins in each treatment (Figure 3C). The greatest number of shared proteins was between samples taken at MP, with 472 of the significantly regulated proteins shared between the extended exposure at 85g/kg and at 105g/kg. The second largest group of overlapping proteins were those which were significantly regulated in all treatments, including 161 proteins. The extended salinity treatments each had many proteins which were only significantly regulated in one treatment, with 144 uniquely significant proteins in the 85g/kg salinity, 110 in the 105g/kg salinity, and 87 in the 75g/kg salinity. A total of 78 proteins were significantly regulated in all four treatments which were above the critical salinity threshold.