Computational protocol: Hepatocytes respond differently to major dietary trans fatty acid isomers, elaidic acid and trans-vaccenic acid

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Protocol publication

[…] The peptides were separated on an Easy-nLC II HPLC system (Thermo Scientific) equipped with a trap column (ReproSil-Pur C18-AQ (5 μm, 2 cm x 100 μm I.D., Thermo Scientific) and an analytical column (ReproSil-Pur C18-AQ column, 3 μm, 10 cm x 75 μm I.D., Thermo Scientific) in-line to a NanoSpray III source (AB Sciex) connected to a TripleTOF 5600 mass spectrometer (AB Sciex) operated under Analyst TF 1.5.1 control. Peptides were eluted at a constant flow of 250 nl/min with a 50 min gradient from 5 to 35 % solvent B (90 % ACN, 0.1 % formic acid) followed by re-equilibration for 10 min back to the starting conditions. Information dependent acquisition was employed acquiring up to 25 MS/MS spectra per cycle using 1.6 s cycle time with an exclusion window of 6 s. [...] All raw MS files were processed using Mascot Distiller 2.5.0 (Matrix Science). The MS data obtained by the analysis of gel lanes were merged into a multi-file-project using the default settings from the ABSciex_5600.opt file except that the MS/MS Peak Picking “Same as MS Peak Picking” was deselected and “Fit method” was set to “Single Peak”. After peak picking all scans, the data were searched against Swiss-Prot Homo Sapiens database (v. 2013_11/12) using Mascot v. 2.3.02 (Matrix Science) []. The Search parameters allowed one missed trypsin cleavage site, propionamide as a fixed modification, and oxidation of methionine as a variable modification. The mass accuracy of the precursor and product ions were set to 10 ppm and 0.2 Da, respectively, the instrument setting was specified as ESI-QUAD-TOF, and a significance threshold of 0.01 was used. The default SILAC R + 6 R + 10 [MD] quantitation protocol was selected using a significance threshold at 0.01, matched rho was 0.7, XIC threshold was 0.1, isolated precursor threshold was set at 0.5 and normalization set to median. Mascot Distiller results were exported to xml files, imported into MS Data Miner v.1.2.0 ( []) and exported into Excel reports. Keratin was excluded as a general contaminant and a fold regulation threshold of 1.3 was selected for significant differentially regulated proteins. Spectra for differentially regulated proteins were manually inspected and proteins were excluded if not at least two unique peptides showed peaks for both light, medium and heavy label significantly above background (ion counts > 50). The data are available via ProteomeXchange with identifier PXD000760 ( Clustering analysis was done using the Functional Annotation tools of DAVID Bioinformatics Resources 6.7 [, ]. […]

Pipeline specifications

Software tools Analyst TF, Mascot Distiller, MS Data Miner, DAVID
Databases UniProt ProteomeXchange
Application MS-based untargeted proteomics
Chemicals Cholesterol, Fatty Acids, Triglycerides, Trans Fatty Acids