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.
The combined .dlib is used as the library for chromatogram library construction in EnclycopeDIA v. 1.12.34. The background is the fasta file with all the protein sequences used to create the Prosit library. The pooled GPF samples (in mzML format) are used to generate an .elib chromatogram library file. This .elib is then used as the library to search the single injection, wide window .mzML files. The quant report resulting from these peptide detections becomes the library in the Skyline document. Search results and single injection .mzML files were imported into Skyline daily v. 22.2.1.256 (MacLean et al., 2010).