Here we report the generation of a data-independent acquisition (DIA) assay library that enables simultaneous targeted proteomics of 1900 O. niloticus gill proteins using a label- and gel-free workflow that is well suited for ecologically relevant field samples. By determining alignment and mismatch between protein and mRNA regulation, the DIA assay library approach generates data that are complimentary rather than redundant to transcriptomics data. Transcript and protein abundance differences in gills of tilapia acclimated to freshwater and brackish water (25 g/kg) revealed non-linearity in salinity-dependent transcriptome versus proteome regulation. Non-linearity was more evident for specific functional groups of genes while other molecular functions/ cellular processes where more highly correlated regarding mRNA and protein regulation. Our study identifies specific salinity-dependent O. niloticus gill functions and processes that rely heavily on mRNA abundance regulation and others that rely more heavily on regulatory mechanisms beyond the transcriptome level. The DIA assay library approach presented here is shown to be a powerful means of complementing transcriptome data with corresponding quantitative proteome data to better discern mechanisms of regulation along the genome to phenome continuum.