Computational protocol: Identification of LEFTY as a molecular marker for ovarian clear cell carcinoma

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[…] Shotgun proteomics using FFPE samples was performed as described previously [, ]. Briefly, 15 μm FFPE sections were deparaffinized, rehydrated, and homogenized in protein extraction buffer (600 mM Tris-HCl pH8.8, 12 mM sodium lauryl sulfate, 12 mM sodium deoxycholate). Samples were incubated at 90°C for 2 h. After cooling to room temperature, reduced disulfides were alkylated by 55 mM iodoacetamide for 0.5 h and enzymatically digested overnight using trypsin (Promega, Madison, WI, USA) and lysyl endopeptidase (Wako Pure Chemical Industries Ltd, Osaka, Japan). The digested samples were acidified with 0.5% trifluoroacetic acid to precipitate surfactants and were desalted with C18-StageTips, followed by lyophilization []. The lyophilized samples were dissolved in 3% acetonitrile, and 0.1% formic acid. The peptides were injected into a trap column: C18 0.1×20 mm (Acclaim PepMap100; Thermo Fisher Scientific, Bremen, Germany), and an analytical column: C18 0.075×120mm (Nano HPLC Capillary Column; Nikkyo Technos, Tokyo, Japan), which was attached to an EASY-nLC 1000 liquid chromatograph (Thermo Fisher Scientific). The flow rate of the mobile phases was 300 nL/min, which consisted of (A) 0.1% formic acid, and (B) 0.1% formic acid and 90% acetonitrile. The mobile phase was programmed as follows: 0-8% B (0-2 min), 8%-32% B (2-132 min), 32-45% B (132-152 min), 45-100% B (152-153 min), and 100% B (153-165 min). Peptides separated by HPLC were introduced to the Q-Exactive mass spectrometer (Thermo Fisher Scientific).The Q Exactive instrument was operated in a data-dependent mode to automatically switch between full scan MS and MS/MS acquisition. Full-scan MS spectra (m/z 350−1500) were acquired in the Orbitrap with a 70,000 resolution at m/z 200 after the accumulation of ions to a 3 × 106 target value. The 10 most intense peaks with a charge state ≥2 from the full scan were selected with an isolation window of 2.0 Da and fragmented in the HCD collision cell with a normalized collision energy of 25%. Tandem mass spectra were acquired in the Orbitrap mass analyzer with a mass resolution of 17,500 at m/z 200 after accumulation of ions to a 2 × 105 target value. The ion selection threshold was 1 × 105 counts, and the maximum allowed ion accumulation times were 60 ms for full MS scans and 60 ms for tandem mass spectra. Typical mass spectrometric conditions were as follows: spray voltage, 2 kV; no sheath and auxiliary gas flow; heated capillary temperature, 250°C; and dynamic exclusion time, 60s.Mass spectral data were processed, exported, and searched against a UniProt human database using SEQUEST by Proteome Discoverer (version 1.3; Thermo Fisher Scientific). Database search parameters were: peptide mass tolerance, 6 ppm; fragment tolerance, 15 ppm; enzyme was set to trypsin, allowing up to two missed cleavages; fixed modifications, carbamidomethyl (cysteine); and variable modifications, oxidation (methionine). The false discovery rate (FDR) was calculated by enabling the peptide sequence analysis using a decoy database. We used a 1% FDR as a cut-off to export results from the analysis. Quantitative analysis of shotgun proteomics data was achieved by spectral counting. […]

Pipeline specifications

Software tools Comet, Proteome Discoverer
Applications MS-based untargeted proteomics, Protein sequence analysis
Chemicals Cisplatin, Formaldehyde, Paraffin