Comprehensive proteomic profiling of early antral follicles from sheep
The present study evaluates the proteome of early antral follicles in Ovis aries. Fifty follicles were collected from ovaries of adult ewes and extracted proteins were trypsin-digested, desalted and analyzed by LC-MS/MS. Peptide-spectrum search was performed using PatternLab V. Genes were screened for potential modulation by miRNAs and protein data, subjected to functional enrichment analysis. Label-free LC-MS/MS allowed the identification of 2,503 proteins in the samples of early antral follicles, confirming vimentin, actin, lamin, heat shock proteins and histones as the most abundant ones. miRNAs modulate the expression of genes involved in cell cycle, cell differentiation, aging, apoptosis, cell death, adipocyte differentiation, cell division. The most important biological processes linked to the follicle proteins were innate immune response, translation, adaptative immune response and protein folding, while molecular functions linked to the proteome of sheep antral follicles related to metal ion binding, ATP binding, oxygen binding, RNA binding and GTP binding, among others. Upload of 2,503 Uniprot accession codes through DAVID platform matched 1,274 genes, associated with translation, metabolic process, proteolysis involved in cellular protein catabolic process, zona pellucida receptor complex and others. KEEG pathways established from the DAVID analysis indicated genes correlated with ovine follicle development, with major pathways listed as carbon metabolism, biosynthesis of amino acids, glutathione metabolism, oxidative phosphorylation, fatty acid degradation and oocyte meiosis. This represents the most comprehensive atlas of proteins expressed in sheep early antral follicles. Data will contribute to future identification of genes and proteins involved in ovine follicular development and oocyte maturation.
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As described in the Methods file, peptides (1 µg) from follicle proteins were analyzed in a chromatographic system with capillary columns (nano-UHPLC Dionex Ultimante 3000) coupled to a hybrid quadrupole-orbitrap mass spectrometer, Orbitrap Q Exactive™ Plus (Thermo Scientific, Germany). Solvents: A (0.1% formic acid) and B (acetonitrile/0.1% formic acid 80/20). Chromatographic system consisted of two columns. An AcclaimTM PepMapTM 100 C18 pre-column (300 µm × 5 mm) with 5 µm-diameter spherical silica particles with 100 Å pore size (Thermo Fisher, USA), with samples injected at 5 µL/min flow rate, with 98% solvent A and 2% solvent B. Then, peptides were separated in a PicoCHIP® analytical column (75µm × 25 cm, 15 µm tip), packed with 1.9 µm Reprosil-PUR C18 particles, 120 Å pore size (New Objective, USA), with elution gradient with solvent B (2-40% during 170 min.; 40-85%; 15 min.; isocratic step of 85% during 5 min.), followed by an isocratic step of 2% solvent B during 19 min. and 30 sec. to re-equilibrate the column. Interface between nanoLC and the hybrid mass spectrometer followed the automatic control of the equipment, using Xcalibur 220.127.116.11 software. Peptide ionization was accomplished in a nanoelectrospray PicoCHIP ion source (New Objective, USA), with spray voltage at 2.5 kV and transfer capillary temperature at 250 °C. MS spectra acquired in positive mode, with DDA cycle (200-2000 m/z range, 140,000 FWHM resolution - Full Width at Half-Maximum) at m/z 400, and with an automatic gain control (AGC) target value of 3 ×106 ions for all FTMS scans and maximum fill time of 45 ms. The survey scan was followed by MS/MS fragmentation by high energy collision dissociation (HCD) of the 20 most abundant precursor ions at each time, under 30% collision energy, with 17,500 FWHM at m/z 400, an AGC target value of 1×106 ions and a minimum value of 8×103 ions for all FTMS scans, and a maximum fill time of 60 ms. Previously fragmented precursor ions were dynamically excluded for 10 s. Peptide-spectrum matching search used Comet search engine (v. 2019.01) in PatternLab V (http://patternlabforproteomics.org), with unreviewed sequences from Ovis aries from UniProtKB (35,607 entries; Sept. 10th, 2021). PatternLab’s Search Database Generator tool included a reverse decoy for target sequence plus sequences from 21 common contaminants. Fixed and variable modifications: carbamidomethylation of cysteine and oxidation of methionine. Validation of peptide-spectrum matches (PSMs) used the Search Engine Processor (SEPro). For each result, XCorr, DeltaCN, DeltaMass, SpecCount Score and Comet’s secondary score values were used to generate a Bayesian discriminator and a cutoff score was established to accept 1% false-discovery rate (FDR). At least 6 amino acid residues were required and the results were filtered to accept PSMs with precursor mass error < 6 ppm. Post-processing filter resulted in FDR < 1% (independent of tryptic status), and ≥2 peptides/identification.