Computational protocol: Deoxycholic Acid Could Induce Apoptosis and Trigger Gastric Carcinogenesis on Gastric Epithelial Cells by Quantitative Proteomic Analysis

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Protocol publication

[…] In this study, a highly specific and sensitive liquid chromatographic/tandem mass spectrometric (LC-MS/MS) method was used to identify differently expressed proteins in GES-1 cells cultured with or without bile acids. The gels were divided into 15 equal pieces according to the proteins' molecular weights. Proteins were then digested with trypsin and the peptides applied to an EASY-nLC system (Proxeon Biosystem, Thermo Fisher Scientific) coupled online to an ESI-LTQ-OrbitrapVelos mass spectrometer (Thermo Fisher Scientific) mostly as described previously []. Peptides were eluted through a trap column and an analytical column packed with C-18 ReproSil 3 μm resin using a gradient from 100% phase A (0.1% formic acid in water) to 35% phase B (0.1% formic acid in acetonitrile) for 150 min. Mass spectra were acquired in a positive mode using the data-dependent automatic (DDA) survey MS scan and tandem mass spectra (MS/MS) acquisition. The DDA survey scan was of the m/z range 350–2000 and resolution 60,000 with a target value of 1 × 10−6 ions. The survey scan was followed by MS/MS of the 15 most intense ions in the LTQ using the collision-induced dissociation (CID), and previously fragmented ions were dynamically excluded for 30 s. Raw data were searched against the Swiss-Prot human proteome database using MaxQuant software (version 1.2.2.5). Searches were performed with the following parameters: tryptic hydrolysis, two missed cleavages, oxidation of methionine as variable modification, carbamidomethylation as fixed modification, and peptide tolerance of 10 ppm. Search results were subsequently processed/filtered through the Search Engine Processor tool [] using a 1% false discovery rate (FDR).Identified proteins were BLAST searched against the NCBI nonredundant database. Ontological analysis of the differentially expressed proteins was performed using the search tool DAVID (http://david.abcc.ncifcrf.gov/). STRING (http://string.embl.de) was used as a database for predicted signaling networks and protein interactions as previously described []. […]

Pipeline specifications

Software tools MaxQuant, SEPro, DAVID
Databases UniProt
Application MS-based untargeted proteomics
Diseases Bile Reflux, Gastritis, Mouth Diseases, Stomach Neoplasms
Chemicals Bile Acids and Salts, Deoxycholic Acid