A better understanding of coho salmon ovarian maturation will inform both aquaculture and conservation efforts. Ovarian maturation is a complex molecular and physiological process resulting in a mature ovum that can be released for spawning and fertilization. The confluence of molecular signals and pathways that must be co-expressed to accomplish full reproductive maturity with high quality oocytes have been previously investigated with with low-resolution proteomics and transcriptomics. This dataset expands proteomics resources for coho salmon by thousands of proteins, allowing for a more in-depth assessment of the range of proteins involved in ovarian maturation and their interactions. We sampled coho salmon at four ovarian developmental stages that encompass the transition from primary to secondary (vitellogenic) growth. Data independent acquisition proteomics was applied to the dataset, resulting in the inference of >5000 proteins in the coho ovarian proteome.

Female coho salmon were reared in a hatchery and sacrificed for ovarian dissection at time points corresponding to the following ovarian stages: early perinucleolar (EPN), late perinucleolar (LPN), early cortical alveolus (ECA), and late cortical alveolus (LCA). Tissues were stored at -80C until processing. Ovarian tissues were homogenized in an ammonium bicarbonate + SDS lysis buffer using a bullet blender. Proteins were extracted and digested using S-trap mini columns from Protifi. Liquid chromatography was run in-line with the mass spectrometer to separate peptides before MS/MS analysis. The fused silica pre-column, with an in-house made kasil frit, was 3.5 cm long with a 150 µm i.d. and the in-house pulled fused silica analytical column was 34 cm long with a 75 µm i.d. Both columns were packed with Dr. Masich C18 3 µm beads in-house. The Lumos mass spectrometer (Thermo Fisher) was run in data independent acquisition (DIA) mode. The pooled samples were analyzed with narrow window (2 m/z) DIA in gas-phase fractions (400-500 m/z, 500-600, 600-700, 700-800, 800-900, 900-1000) to generate an in-depth spectral library. Individual ovary peptide samples were analyzed with wider window DIA (8 m/z) across the m/z range of X-Y. Sample order was randomized for mass spectrometry analysis and between each 3-4 samples a QC (PRTC + BSA) and a solvent blank were run. Mass spectrometry results were analyzed using the Prosit-EncyclopeDIA-Skyline pipeline described in Searle et al. (2020). Raw mass spectrometry files were converted to .mzML format in MSConvert with the following parameters: remove titleMaker filter, write index, TPP compatibility, use zlib compression, SIM as spectra, and added filters with default settings (peakPicking and demultiplex). The database used for peptide detection was the predicted coho proteome (GCF_002021735.2_Okis_V2.protein.faa). We discovered the some proteins of interest for ovarian maturation were not included in this proteome, so we added sequences for follicle stimulating hormone (FSH), luteinizing hormone (LH), the receptors for FSH and LH, two 5-alpha reductases, and an ovarian aromatase. All sequences were derived from coho salmon, except for the FSH receptor, which came from the Atlantic salmon proteome. The coho proteome was converted to a prosit csv in EnclycopeDIA v. 1.4.10 and the .csv was subdivided into five sub-proteomes since Prosit has a file size limit. Prosit creates theoretical spectral libraries that frequently improve peptide detections in our pipeline. Each prosit .txt file was downloaded and converted to .dlib format in EncyclopeDIA v.1.4.10. The five .dlibs were combined into a single file for further analyses.

coho salmon ovaries