DOI: 10.25345/c5hp1f
title: MassIVE MSV000088230 - Dietary relevant concentrations of BDE-47, -99, -209 and their mixture differently affect HepG2 transcriptomic and proteomic profiles.
title: Polybrominated diphenyl ethers (PBDEs) are man made chemicals with flame retardant properties which are added to commercial products and materials such as textiles, plastics, electronic devices, household products and furniture to reduce their flammability. In the present study we investigated the differences in the mode of action of three congeners more abundant in the diet yielded relative toxicities scores BDE-209 , 47 , 99 considering their ternary mixture, also, at the mean concentration found in food, which is not cytotoxic, 1nM, by treating the human hepatoma cell line (HepG2) and analyzing correspondent transcriptomic and proteomics profiles. HepG2 cells, treated with 1nM of each congener and also with a mixture containing the three congeners together, were examined with a shotgun proteomic approach in order to analyze the protein differential expression between treated samples, considering also the background proteins coming from control samples represented by untreated HeG2 cells. We consider two biological replicate and two technical replicate for each treated sample, including control samples. HepG2 cell pellets dedicated to proteomics analysis were homogenized in RIPA buffer (50mM Tris-HCl, pH 7.5; 150mM NaCl; 1% Triton X 100; 1% sodium deoxycholate; 0.1% SDS) added with a protease inhibitor cocktail (Sigma-Aldrich) on ice. Protein lysates were clarified by centrifugation at 14,000 rpm for 15 min at 4 C. Total protein content in each sample was determined by the Pierce BCA Protein Assay Kit (Thermo scientific, Rockford, IL, USA) according to manufacturers instructions, reading absorbance at 562 nm on a NanoDrop spectrophotometer (Thermo). 25 ug of total proteins from each sample were loaded and separated on a 1D gel NuPAGE 4 12% (Novex, Invitrogen, Carlsbad, CA, USA) run in MOPS buffer and stained with the Colloidal Blue Staining kit (Invitrogen, Carlsbad, CA, USA). The entire gel lanes were cut in 12 homogeneous slices which were subjected to trypsin digestion after the reduction and alkylation step, according to an established protocol (Snevechenko et al. 1996). The resulting peptide mixtures were separated by a nanoflow reversed-phase liquid chromatography tandem mass spectrometry using an HPLC Ultimate 3000 (DIONEX, Sunnyvale, CA, USA) connected with a linear ion trap (LTQ XL, ThermoElectron, San Jose, CA) equipped with a nano-electrospray ion source (ESI). Peptides were at first desalted in a trap column (Acclaim PepMap 100 C18, LC Packings, DIONEX) and then separated in a 10 cm long reverse phase column (Silica Tips FS 360-75-8, New Objective, Woburn, MA, USA) slurry packed in-house with 5 um, 200 A pore size C18 resin (Michrom BioResources, CA). Peptides were eluted using a linear gradient from 96% of buffer A (H2O with 5% acetonitrile and 0.1% formic acid) to 60% of buffer B (acetonitrile with 5% H2O and 0.1% formic acid) for 40 min, at 300 nL/min flow rate. Peptide ions were analyzed in positive mode, and the HV potential was set up around 1.7 kV. Full MS spectra (m/z 400-2000 mass range) were acquired in a data dependent mode in which each full MS scan was followed by five MS/MS scans, where the five most abundant molecular ions were dynamically selected and fragmented by CID, using a normalized collision energy of 35%. Database searches were performed using the Sequest algorithm, embedded in Proteome Discoverer 1.4 (PD1.4, Thermo Fisher Scientific). The search criteria for protein identification were set to at least two matching peptides per protein. Tandem mass spectra were analyzed by Sequest HT and matched against the human database (UniProtKB/Swiss Prot protein database, featuring 23.316 entries). Spectra files were searched using Proteome Discoverer 1.4 (ThermoFisher) as search engine with carbamidomethylation on cysteine as static modification, methionine oxidation as variable one and full tryptic peptides with a maximum of two missed cleavages allowed were considered for identification. A mass tolerance of 1.5 Da for precursor ion and 0.8 Da for fragment ions were applied. The Percolator node of PD1.4 was used for peptide validation based on q-values, choosing high confidence corresponding to a false discovery rate (FDR) inferior to 1% on peptide level. Proteins were identified with a minimum of 2 peptides. Two biological replicas were analyzed and for each replica, two technical replicas were performed.