Plasma proteins of marine (Bodega Harbor) and landlocked freshwater (FW, Lake Solano) three-spined sticklebacks were quantified by targeted proteomics. Proteins to be targeted were selected based on a PEAKS label-free quantitative survey of the entire identified plasma proteome. Forty-five plasma proteins were confirmed as highly population-specific with regard to their abundance in stickleback plasma. Proteins with functions in cell adhesion, tissue remodeling, proteolytic processing, defense signaling, and bone morphogenesis are more abundant in marine sticklebacks. Proteins involved in translation, heme biosynthesis, and lipid transport, which may be localized in plasma microparticles are more abundant in FW sticklebacks. Several uncharacterized proteins with unknown functions also show highly population-specific abundance profiles in three-spined stickleback plasma. The molecular phenotypes identified in this study provide insight into targets of selection and molecular mechanisms of adaptation during FW invasion of three-spined sticklebacks.

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 (34 g/kg salinity SW) and by unbaited minnow traps from Lake Solano (FW) in August 2014 using California Department of Fish and Wildlife scientific collecting permit SCP12637. They were sacrificed under protocols approved by the University of California Davis (IACUC number 18010, AALAC number A3433-01) and blood collected from the dorsal aorta using EDTA-coated hematocrit capillary tubes (Thermo Fisher, Waltham, MA, USA) within 2 h of capture. Plasma was prepared by centrifugation of blood at 2000×g, 4_C for 10 min followed by collection of supernatant and storage at −80_C until processed for protein extraction (e.g. from exosomes), protein assay, and in solution trypsin digestion 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.