A proteomic landscape of diffuse-type gastric cancer
Here we present a dataset from 84 DGC patients with paired tumor and nearby tissue. Tumors and their nearby tissues were evaluated by pathologists. Nearby tissues were designated as non-cancerous and were greater than 5 cm away from the surgery margin. Hematoxylin and eosin (H&E) stained sections were examined by two expert gastrointestinal pathologists (Z.L. and Yumei Lai) independently to confirm: (1) diffuse type (Lauren type); (2) >50% tumor cell nuclei; (3) <20% necrosis in tumor tissue; (4) no tumor cells in nearby tissue.
Sample Processing Protocol
Specimens in dry ice were transferred to Beijing Proteome Research Center within three hours after surgery. Samples were minced and lysed in lysis buffer (8M Urea, 100mM Tris Hydrochloride, pH8.0) containing protease and phosphatase Inhibitors (Thermo Scientific) followed by 1 min sonication (3s on and 3s off, amplitude 25%). The lysate was centrifuged at 14,000g for 10 min and the supernatant was collected as whole tissue extract. Protein concentration was determined by Bradford protein assay. Extracts from each sample (100 μg protein) was reduced with 10 mM dithiothreitol (DTT) at 56°C for 30 min and alkylated with 10 mM iodoacetamide (IAA) at room temperature in the dark for additional 30 min. Samples were then digested using the FASP method54 with trypsin; tryptic peptides were separated in a home-made reverse-phase C18 column in a pipet tip. Peptides were eluted and separated into nine fractions using a stepwise gradient of increasing acetonitrile (6%, 9%, 12%, 15%, 18%, 21%, 25%, 30%, and 35%) at pH 10. The nine fractions were combined to six fractions, dried in a vacuum concentrator (Thermo Scientific), and then analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Samples were analyzed on Orbitrap Fusion, Orbitrap Fusion Lumos and Q Exactive Plus mass spectrometers (Thermo Fisher Scientific, Rockford, IL, USA) coupled with an Easy-nLC 1000 nanoflow LC system (Thermo Fisher Scientific), or a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Rockford, IL, USA) connected to an UltiMate 3000 RSLCnano System (Thermo Fisher Scientific). Dried peptide samples were re-dissolved in Solvent A (0.1% formic acid in water) and loaded to a trap column (100 μm × 2 cm, homemade; particle size, 3 μm; pore size, 120 Å; SunChrom, USA) 569 with a max pressure of 280 bar using Solvent A, then separated on a home-made 150 μm × 12 cm silica microcolumn (particle size, 1.9 μm; pore size, 120 Å; SunChrom, USA) with a gradient of 5-35% mobile phase B (acetonitrile and 0.1% formic acid) at a flow rate of 600 nl/min for 75 min. The MS analysis for QE HF and QE Plus were performed with one full scan (300-1400 m/z, R=60,000 at 200 m/z) at automatic gain control target of 3e6 ions, followed by up to 20 data-dependent MS/MS scans with higher-energy collision dissociation (target 2×103 ions, max injection time 40 ms, isolation window 1.6 m/z, normalized collision energy of 27%), detected in the Orbitrap (R=15,000 at 200 m/z). For detection with Fusion or Fusion Lumos mass spectrometry, a precursor scan was carried out in the Orbitrap by scanning m/z 300 -1400 with a resolution of 120,000 at 200 m/z. The most intense ions selected under top-speed mode were isolated in Quadrupole with a 1.6 m/z window and fragmented by HCD with normalized collision energy of 35%, then measured in the linear ion trap using the rapid ion trap scan rate. Automatic gain control targets were 5×105 ions with a max injection time of 50 ms for full scans and 5×103 with 35 ms for MS/MS scans. Dynamic exclusion time was set as 18s. Data were acquired using the Xcalibur software (Thermo Scientific).
Data Processing Protocol
Raw files were searched against the human National Center for Biotechnology Information (NCBI) Refseq protein database (updated on 04-07-2013, 32015 entries) by Mascot 2.5 (Matrix Science Inc). The mass tolerances were 20 ppm for precursor and 50 mmu for product ions from Q Exactive Plus and Q-Exactive HF, and 20 ppm for precursor and 0.5 Da for product ions for Fusion and Q-Exactive HF, respectively. Up to two missed cleavages were allowed. The search engine set cysteine carbamidomethylation as a fixed modification and N-acetylation, oxidation of methionine as variable modifications. Precursor ion score charges were limited to +2, +3, and +4. The data were also searched against a decoy database so that protein identifications were accepted at a false discovery rate of 1%.