A data-independent acquisition (DIA) assay library for quantitation of environmental effects on the kidney proteome of Oreochromis niloticus
- Organism: Oreochromis niloticus
- Instrument: impact II
Nile tilapia, Kidney, DIA-LCMS2, Environmental salinity, Bottom-up proteomics, Label-free quantitative proteomics
- Lab head:
Here we report the generation of a data-independent acquisition (DIA) assay library that enable simultaneous targeted proteomics of thousands of O. niloticus kidney proteins using a label- and gel-free workflow that is well suited for ecologically relevant field samples. We demonstrate the usefulness of these libraries by discerning environmental effects on the kidney proteome of O. niloticus. Moreover, we demonstrate that the DIA assay library approach generates data that are complimentary rather than redundant to transcriptomics data. Transcript and protein abundance differences in kidneys of tilapia acclimated to freshwater and brackish water (25 g/kg) were correlated for 2114 unique genes. A high degree of non-linearity in salinity-dependent regulation of transcriptomes and proteomes was revealed suggesting that the regulation of O. niloticus renal function by environmental salinity relies heavily on post-transcriptional mechanisms.
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. Kidney 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 kidneys 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.
Created on 9/1/20, 12:37 AM