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.

O. niloticus were raised in freshwater (FW, <1 g/kg) and randomly distributed into two groups: brackish water (BW, 25 g/kg) and FW handling controls. The BW group was exposed to a gradual salinity increase of 5 g/kg per day up to a final salinity of 25 g/kg. Feeding occurred (ad libitum) was ceased 24 hours before sample collection. After 24 hours at the final salinity, fish were sacrificed and immediately dissected. Gill epithelium samples were removed and divided into two aliquots, which were immediately snap-frozen in liquid nitrogen and stored at -80°C. One aliquot from each individual was subsequently processed for proteomics, the other was processed for RNAseq.

Samples represent protein extracts from gill epithelium of Oreochromis niloticus. Each sample is a biological replicate obtained from a different fish. Six biological replicates from FW-acclimated fish and six biological replicates from BW-acclimated fish were analyzed with DDA-LCMS2 to annotate MS2 spectra for raw spectral library construction. The same samples were also processed with DIA-LCMS2 and used as training samples for filtering the raw spectral library to yield the final DIA assay library.