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Dietary relevant concentrations of BDE-47, -99, -209 and their mixture differently affect HepG2 transcriptomic and proteomic profiles.

ABSTRACT: 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.

INSTRUMENT(S): LTQ XL ETD

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Marialuisa Casella

PROVIDER: MSV000088230 | MassIVE | Thu Oct 14 05:46:00 BST 2021

SECONDARY ACCESSION(S): PXD029124

REPOSITORIES: MassIVE

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Project description:The following cell lines were used for this study: Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs) derived from 5 human (Coriell YRI, NIGMS Human Genetic Cell Repository, GM18505, GM18507, GM18516, GM19193, GM19204), and 5 chimpanzee (Pan troglodytes) individuals (New Iberia Research Center: Min 18358, Min 18359; Coriell/IPBIR: NS03659, NS04973, Arizona State University, Pt91), and rhesus Herpesvirus papio transformed LCLs from 5 rhesus macaque (Macaca mulatta) individuals (Harvard Medical School, NEPRC: 150-99, R181-96, R249-97, 265-95, R290-96). Cells were maintained at identical conditions of 37° with 5% CO2 in RPMI media with 15% FBS, supplemented with 2 mM L-glutamate, 100 IU/ml penicillin, and 100 ug/ml streptomycin. Internal standard LCL (Coriell YRI, NIGMS Human Genetic Cell Repository, GM19238) was grown in RPMI minus L-Lysine and L-Arginine, 15% dialyzed FBS, and L-13C615N4-arginine (Arg-10) and L-13C615N2-lysine (Lys-8) (Cambridge Isotopes, Andover, MA, USA) supplemented with 2 mM L-glutamate, 100 IU/ml penicillin, and 100 ug/ml streptomycin under identical conditions as the unlabeled LCLs. The internal standard LCL was grown for 6 doublings to assure complete SILAC label incorporation. Complete label incorporation was verified by analyzing the protein lysate from the labeled LCL alone by high-resolution LC-MS/MS. LCLs were washed in PBS three times and then lysed using the UPX Universal Protein Extraction Kit (Expedeon Inc., San Diego, CA, USA). Protein quantitation was performed using the Qubit fluorometry assay (Invitrogen, Carlsbad California, USA) and the reducing agent-compatible (RAC) version of the BCA Protein Assay (Thermo Scientific, Waltham, Massachusetts, USA). 12ug of each sample was combined with 12ug of the SILAC labeled lysate from human LCL GM19238. Note that the SILAC lysate was prepared once and used as an internal standard through the quantification of the 15 cell lines. 24ug of each combined sample was then processed by SDS-PAGE using a 4-12% Bis Tris NuPage mini-gel (Invitrogen, Carlsbad California, USA). Calibration was with Thermo PageRuler broad range markers. Each of 40 gel segments were processed by in-gel digestion using a ProGest robot (DigiLab, Marlborough, MA, USA) with the following protocol: wash with 25mM ammonium bicarbonate followed by acetonitrile, reduce with 10mM dithiothreitol at 60°C followed by alkylation with 50mM iodoacetamide at room temperature, digest with trypsin (Promega, Madison, WI, USA) at 37°C for 4h, and quench with formic acid. The supernatant was analyzed directly without further processing. Each of gel digest was analyzed by nano-LC/MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher LTQ-Orbitrap Velos Pro. Peptides were loaded on a trapping column and eluted over a 75um analytical column at 350nL/min using a 1-hour LC gradient. Both columns were packed with Jupiter Proteo resin (Phenomenex, Torrance, California, USA). The mass spectrometer was operated in data-dependent mode, with MS performed in the Orbitrap at 60,000 FWHM resolution and MS/MS performed in the LTQ. The fifteen most abundant ions were selected for MS/MS. Low-level data analysis was performed using the open-source proteomics software tool PVIEW (Release December 23, 2012; http://compbio.cs.princeton.edu/pview). As input to PVIEW, we generated in silico translations of coding genes from the UCSC Genome Browser database based on gene models from build hg19 of the human genome. Each protein sequence entry retained the corresponding Ensembl gene identifier and gene symbol. Database searches were performed using +-4 p.p.m. MS1 tolerance and an MS2 window tolerance of +-0.5 Da. Up to 2 missed tryptic cleavages were allowed during search. Carboxyamidomethylation of cysteine was used as fixed modification. Up to two methionine oxidations were allowed as variable modifications of a tryptic peptide. Peptide spectrum matches were obtained at a stringent false discovery rate (FDR) of 1%. We used the median log2(sample/standard) ratio across all independent quantifications of a protein (distinct peptides including duplicate peptide measurements across fractions and for differing charge states). Note that use of the hg19 database was sufficient as SILAC pairs as the expected isotope shift used for quantification were only present for peptides that that had the same underlying sequence in the human internal standard line. Associated RNA-seq data have been deposited to GEO with accession number GSE49682.

Project description:Four bile samples collected from patients with a malignant (PAC, CC) or a nonmalignant (CP, BS) biliary stenosis were used for comparative proteomic analysis. Briefly, bile samples were centrifuged at 16,000/g/ for 10 min at 4 C. Each supernatant was delipided with Cleanascite (Biotech Support Group, North Brunswick, NJ, USA) and ultrafiltrated using a 3 kDa filter cut-off (YM-3 centricon, Millipore, Bedford, MA, USA). For each sample, 50 ug of proteins were mixed with 0.5 M triethylammonium bicarbonate (TEAB) buffer pH 8.0 to a total volume of 100 uL. One ug of bovine lactoglobulin (LACB) was spiked in each sample as an internal standard. Proteins were reduced and alkylated before digestion of N-linked oligosaccharides from glycoproteins using PNGase F (Sigma-Aldrich, St. Louis, MO, USA). Proteins were then digested with trypsin and the resulting peptides were tagged with one of the isobaric tags from the iTRAQ reagents 4plex kit (AB Sciex, Foster City, CA, USA) according to the manufacturer's instructions. The four samples were then pooled and dried under vacuum. Labeled peptides were fractionated according to their isoelectric point on an Agilent 3100 OFFGEL fractionator using 24 cm pH 3-10 linear immobilized pH gradients (GE Healthcare, Chalfont St. Giles, UK) following manufacturer's instructions. Fractions were then recovered in separate tubes for high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). MS analysis was performed on a LTQ Orbitrap XL from Thermo Electron (San Jose, CA, USA) equipped with a NanoAcquity HPLC system from Waters. Peptides were trapped on a home-made 5 um 200 A Magic C18 AQ (Michrom, Auburn, CA, USA) 0.1 x 20 mm pre-column and separated on a home-made 5 um 100 A Magic C18 AQ (Michrom) 0.75 x 150 mm column with a gravity-pulled emitter. The analytical separation was run for 65 min using a gradient of H_2 O/formic acid (FA) 99.9%/0.1% (solvent A) and CH_3 CN/FA 99.9%/0.1% (solvent B). The gradient was run at a flow rate of 220 nL/min as follows: 5% B for 1 min, from 5 to 35% B in 54 min, from 35 to 80% B in 10 min. For MS survey scans, the OT resolution was set to 60,000 and the ion population was set to 5 x 10^5 with an m/z window from 400 to 2000. Maximum of 3 precursors were selected for both collision-induced dissociation (CID) in the LTQ and high-energy C-trap dissociation (HCD) with analysis in the Orbitrap (OT). For MS/MS in the LTQ, the ion population was set to 1 x 10^4 (isolation width of 2 m/z) while for MS/MS detection in the OT, it was set to 2 x 10^5 , with resolution of 7,500, first mass at m/z = 100, and maximum injection time of 750 ms. The normalized collision energies were set to 35% for CID and 60% for HCD. Peak lists from MS analysis were searched against Uniprot_Swissprot database (version 2011_2) using Phenyx protein identification software (GeneBio, Geneva, Switzerland). /Homo Sapiens/ taxonomy was specified for database searching. The parent ion tolerance was set to 10 ppm. Variable amino acid modifications were oxidized methionine and deamidated asparagine. Carbamidomethylation of cysteines and iTRAQ-labeled peptides on the amino terminus and lysine were set as fixed modifications. Trypsin was selected as the enzyme, with one potential missed cleavage, and the normal cleavage mode was used. Only one search round was used with selection of "turbo" scoring. The peptide p value was 10^-2 for LTQ-OT data. Protein and peptide scores were set up to maintain the false positive rate below 5%.

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Dietary relevant concentrations of BDE-47, -99, -209 and their mixture differently affect HepG2 transcriptomic and proteomic profiles.

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