Sample processing for proteomics
Samples from seven treatments were used to compare proteome changes based on time and salinity level, including eight samples each from 14-day acclimations to 85g/kg and 105g/kg as well as the FW control and the 12-week 75g/kg treatment and FW control, and six samples from the extended constant salinity to MP at 85g/kg and 105g/kg due to experimental deaths of two fish in each treatment. Extraction and in-solution trypsin digestion of proteins were performed as previously reported (Root et al., 2021a). Peptide samples (2 µl, 100 ng/µl) were injected with a nanoAcquity sample manager, trapped for 1 min at 15 µL/min on a Symmetry trap column, and separated on a 1.7µm particle size BEH C18 column by reversed phase liquid chromatography using a nanoAcquity binary solvent manager. Peptides were eluted using a 125 min linear gradient ranging from 3% to 35% acetonitrile (ACN) directly (online) into a UHR-qTOF mass spectrometer using a pico-emitter tip. Batch processing of samples was controlled with Hystar 4.1 and a 68 fmol BSA peptide mix quality control standard was used intermittently between samples to monitor instrument performance.