Targeted proteomics was performed to validate differences in renal proteomes of a marine (Bodega Harbor) compared to a freshwater (FW, Lake Solano) population of three-spined sticklebacks (Gasterosteus aculeatus). Twentysix protein targets were selected based on prior unbiased quantitative MS1 profiling with PEAKSQ. Half (13) of these targets are overrepresented and the other half are underrepresented in kidneys of marine compared to FW fish. Label-free data-independent acquisition (DIA) and subsequent quantitative Skyline analysis were used for targeted proteomics to validate relative protein abundance differences in kidneys of marine and FW sticklebacks. The results of this experiment show that glyoxylate/ amino acid metabolism, organic osmolyte (trimethylamine oxide, urea, betaine, sorbitol) synthesis, and glutathione metabolism are significantly elevated in kidneys of marine fish. Conversely, proteins involved in actin polymerization, cell adhesion, calcium signaling, protein turnover, and energy metabolism are significantly more abundant in kidneys of FW fish.

Tryptic peptides from each sample (200 ng on column) were injected with a nanoAcquity sample manager (Waters), trapped for 1 min at 15 µL/min on a Symmetry trap column (Waters 186003514), and separated on a 1.7µm particle size BEH C18 column (250mm x 75µm, Waters) by reversed phase LC using a nanoAcquity binary solvent manager (Waters). A 125 min linear gradient ranging from 3% to 35% acetonitrile was used. Peptides were ionized by nano-ESI using a pico-emitter tip (New Objective) and analyzed by an ImpactHD UHR-QTOF mass spectrometer (Bruker). Batch-processing of samples was controlled with Hystar 3.2 (Bruker). The mass range was set to 490 – 910 m/z at 25 Hz scan rate with an isolation width of 11 m/z (1 m/z overlap). Spectral libraries were created from prior precursor-dependent MS1 data (< 20 ppm mass error) that were analyzed by unbiased PEAKSQ quantitative profiling (BSI) to identify protein targets that differ in abundance between stickleback populations. DIA data for targeted proteins were statistically analyzed using Skyline 3.1 (MacCossLab, UW).

Adult sticklebacks were collected by seining from Bodega Harbor, California, and by unbaited minnow traps from Lake Solano, California, in August 2014 using California Department of Fish and Wildlife scientific collecting permit SCP12637. Six males and six females from each population were obtained. Fish were sacrificed under protocols approved by the University of California Davis (IACUC number 18010, AALAC number A3433-01) within 2h of capture. Kidneys were dissected on ice and immediately snap-frozen in liquid nitrogen. Protein extraction from stickleback kidneys, protein assay, and in solution digestion with immobilized trypsin were performed as previously described [1]. 1. Kültz D. et al. (2013) Quantitative molecular phenotyping of gill remodeling in a cichlid fish responding to salinity stress. Molecular & Cellular Proteomics 12 (12), 3962-3975.