Experimental design

The rat tissues analysed in this study were obtained from animals as previously described18. Briefly, the experimental protocol was as follows. Following 20 days of acclimatization, Harlan Sprague-Dawley rats at 5 weeks of age were randomly assigned on a weight basis into groups of 10 animals. Animals were fed with the standard diet A04 (Safe, France) including 33% maize DKC 2675 over two years. All feed formulations consisted of a balanced diet, chemically measured as substantially equivalent. All animals were kept in polycarbonate cages (820 cm2, Genestil, France). The location of each cage within the experimental room was regularly changed. The litter (Toplit classic, Safe, France) was replaced twice weekly. The animals were maintained at 22 ± 3 °C under controlled humidity (45% to 65%) and air purity with a 12 h-light/dark cycle, with free access to food and water. All reagents used were of analytical grade. The animal experimental protocol was conducted in accordance with the regulations of the local ethics committee in an animal care unit authorized by the French Ministries of Agriculture and Research (Agreement Number A35-288-1). Animal experiments were performed according to ethical guidelines of animal experimentation (regulation CEE 86/609).

Groups of 10 animals had access to either plain water (control) or to the same water supplemented with 1.1 × 10−8% of Roundup (0.1 ppb or 0.05 μg/L glyphosate equivalent dilution). The commercial formulation of Roundup used was Grand Travaux Plus (450 g/L glyphosate, approval 2020448; Monsanto, Belgium). The required level of Roundup dilution in drinking water was confirmed by measurement of glyphosate concentration by HPLC-MS/MS. Similarly, glyphosate stability in solution was studied and validated during the 7 day period between two preparations of the test treatment solutions.

Triglycerides were quantified by the enzymatic reactions of Fossati and Trinder45. The procedure measures the concentrations of total triglycerides by converting them to glycerol and free fatty acids by lipase. The glycerol is then converted to glycerol-3-phosphate and finally to hydrogen peroxide. A colored complex is formed from hydrogen peroxide, 4-aminophenazone and 4-chlorophenol via an enzymatic reaction with a peroxidase. The absorbance of the complex is measured at 510 nm.

Tissue sampling

Animals were sacrificed at the same time of day during the course of the study either to comply with animal welfare regulations to avoid unnecessary suffering (for example, resulting from 25% body weight loss, presence of tumours over 25% bodyweight, hemorrhagic bleeding, or prostration) or at the termination of the study period of 2 years. Animals were sacrificed by exsanguination under isoflurane anesthesia. Livers were divided in two and half snap frozen in liquid nitrogen/dry ice and stored at −80 °C.

Proteome profiling using Tandem Mass Tag-LC-MS/MS

Transverse cross sectional slices of liver were lysed in 8 M lysis buffer (urea, NaCl, Tri-HCl, dH2O, phosphatase and protease inhibitor) and the protein concentration of the resulting homogenate calculated using a Nanodrop protein assay (used on the A280 setting). Samples were reduced with 5 mM dithiotreitol (Sigma, UK), alkylated by treatment with 14 mM iodoacetamide (Sigma) and digested with 12 μg bovine sequencing grade trypsin (Roche, Germany, Ref. 11418475001) at 37 °C for 18 hours. Subsequently, peptides were purified and extracted using Waters Sep-Pak Vac 3cc 200 mg tC18 cartridges (Waters, WAT054925) in accordance to with the manufacturer’s instructions before each sample was labelled by incubation with 60 mM TMT10plex Isobaric Label Reagents (Thermo-Scientific, ref 90406). Labelled peptides were then purified and extracted again with the Waters Sep-Pak Vac 3cc 200 mg tC18 cartridge, before being fractionated by strong cation exchange (SCX) across an increasing salt concentration using elution buffers containing different concentrations of KCl: ranging from 0 mM KCl in the first fraction to 350 mM KCl in the 10th fraction. A 1/10th aliquot of the eluted peptide fractions were separated and lyophilised for direct analysis by LC-MS/MS. The remaining 9/10th of column eluate was enriched for phosphopeptides using a Pierce TiO 2 Phosphopeptide Enrichment and Clean-up Kit (Pierce, Prod # 88301).

Un-enriched samples were re-suspended in 100 μl of 50 mM ammonium bicarbonate and F1&6, F2&7, F3&8, F4&9 and F5&10 pooled to give 5 fractions. Phosphopeptide enriched samples were re-suspended in 30 μl 50 mM ammonium bicarbonate and pooled in the same way as the un-enriched samples to also give 5 fractions. Both enriched (8 μl) and un-enriched (5 μl) fractions were loaded onto a 50 cm EASY-spray column (Thermo Scientific) and quantitative analysis was performed using the Orbitrap Velos-Pro mass spectrometer (Thermo Scientific) in positive ion mode. The peptides and phosphopeptides were separated by gradient elution, from 5–80% 0.1% trifluoroacetic acid in acetonitrile (5–40% from 0–100 minutes, 40–80% from 100–110 minutes), at a flow rate of 300 nl/min. Mass spectra (m/z) ranging from 400–1600 Daltons was acquired at a resolution of 60,000 and the 10 most intense ions were subjected to MS/MS by HCD fragmentation with 35% collision energy.

Protein identification was performed with Proteome Discoverer 1.4 (Thermo Fisher Scientific Inc.). Raw files were imported and searched against the UniProtKB/Swiss-Prot Database using Sequest for Proteome Discoverer. Exported raw data for the two TMT10plex sets is available as Supplementary Tables 1 and 2. Raw files for both enriched and un-enriched fractions were merged together in a single file search for each of the two TMT10plex sets. Precursor mass tolerance for the searches was set at 20ppm and fragment mass tolerance at 0.8ppm. The taxonomy selected was Rattus norvegicus and three enzymatic miscleavages were allowed. Dynamic modifications selected on the search were Oxidation/+15.995 Da (M), Phospho/+79.966 Da (S, T, Y) and Deamidated/+0.984 (N, Q) and static modifications were Carbamidomethyl/+57.021 Da (C), TMT10plex/229.163 Da (K), TMT10plex/229.163 Da (Any N-terminus).

Proteome verification using Tandem Mass Tag - selected reaction monitoring experiment

Samples were prepared in the same way as the discovery experiment up to the TMT labelling stage. For selected reaction monitoring (SRM) analysis the twenty individually TMT10plex Heavy labelled samples were not combined but analysed individually against a pool of the twenty liver samples labelled with TMTZero/Light Label Reagent (Thermo-Scientific) included as a single point reference sample. All labelled peptides were purified and extracted before being lyophilised prior to mass spectrometry analysis.

Peptides were selected for TMT-SRM verification based on findings from the discovery analysis. Peptides chosen were significantly regulated against parameters where peptides had to display a fold change ≥1.2, either up or down-regulated, whilst being significant to a p-value of ≤0.01. Peptides were re-solubilised in 2% acetonitrile/0.1% trifluoroacetic acid and 2.5 μg of protein loaded onto a 50 cm EASY-Spray column (Thermo Scientific) using initial gradient conditions identical to those used in the discovery experiment. The LC system was coupled to a TSQ Vantage mass spectrometer (Thermo-Scientific) set in positive ion mode with Q1 and Q3 peak width settings of 1 full width at half its maximum height (FWHM). Capillary temperature (°C), collision gas pressure (mTorr) and spray voltage (V) were set at 270, 1.2 and 1800 respectively. In order to asses peptide transition specificity and abundance and define retention times, initial optimisation was performed using the reference sample alone. Following this a combined 1:1 mix with a random Heavy TMT labelled sample was then measured. The final method contained all successful transitions for both TMT Heavy and TMT Light versions of the peptides (Supplementary Table 4; 68 transitions from 10 peptides and 9 proteins). This final method was applied in three separate injections to each Heavy labelled experimental sample, which had been mixed in a 1:1 ratio with the Light labelled reference sample. Peptides were separated over 35 minutes, using gradient elution 5–80% 0.1% trifluoroacetic acid in acetonitrile (0–5% from 0–3 minutes, 5–50% from 3–7 minutes, 50–65% from 7–30 minutes and 65–80% from 30–35 minutes) at a flow rate of 300 nl/min.

Data was analysed using Skyline software46, with all peak matching also being visually verified. Peak area ratios between Light and Heavy transitions were generated for each sample and exported into Excel. When importing raw SRM data files from the TSQ vantage into Skyline software, a unique peak picking algorithm was used to confidently and accurately assign the best transition peaks for each peptide. Peptides were only taken forward for analysis if all assigned peaks were within a similar retention time period across all 60 samples. From the assigned peaks Skyline software was then used to produce a ‘total ratio’ between the Light and Heavy transitions for all of the 60 raw data files (3 × 10 control sample injection and 3 × 10 treated sample injections). This ‘total ratio’ value is a mean of the transition ratios, where the ratio is the comparison of the heavy transition peak areas to the light transition peak areas. The total ratio was averaged across all control and treated samples and a fold change between the two calculated. Coefficient of variation (CV) values between technical sample repeats were calculated and also averaged across control and treatment samples. Any peptides which displayed an average technical CV value higher than 15% were excluded from further analysis.

Metabolome analysis

Semiquantitative metabolomics analysis was performed by ultra-high performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) and gas chromatography-mass spectroscopy (GC-MS) at Metabolon Inc. (Durham, NC, USA)47.

Samples were prepared using the automated MicroLab STAR® system from Hamilton Company (Reno, NV, USA). A recovery standard was added prior to the first step in the extraction process for QC purposes. In order to remove protein, dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder, 2000) followed by centrifugation. The resulting extract was divided into five fractions: one for analysis by UPLC-MS/MS with positive ion mode electrospray ionization, one for analysis by UPLC-MS/MS with negative ion mode electrospray ionization, one for LC polar platform, one for analysis by GC-MS, and one sample was reserved for backup. Samples were placed briefly on a TurboVap® (SOTAX Corp, Westborough, MA, USA) to remove the organic solvent. For LC, the samples were stored overnight under nitrogen before preparation for analysis. For GC, each sample was dried under vacuum overnight before preparation for analysis.

The LC-MS portion of the platform was based on a Waters ACQUITY ultra-performance liquid chromatography (UPLC) system (Waters Corp, Milford, MA, USA) and a ThermoFisher Scientific Q-Exactive high resolution/accurate mass orbitrap mass spectrometer operated at a 35,000 mass resolution, which was interfaced with a heated electrospray ionization (HESI) source. The sample extract was dried then reconstituted in acidic or basic LC-compatible solvents, each of which contained 12 or more injection standards at fixed concentrations to ensure injection and chromatographic consistency. One aliquot was analyzed using acidic positive ion-optimized conditions and the other using basic negative ion-optimized conditions in two independent injections using separate dedicated columns (Waters UPLC BEH C18-2.1 × 100 mm, 1.7 μm). Extracts reconstituted in acidic conditions were gradient eluted using water and methanol containing 0.1% formic acid, while the basic extracts, which also used water/methanol, contained 6.5 mM ammonium bicarbonate. A third aliquot was analyzed via negative ionization following elution from a HILIC column (Waters UPLC BEH Amide 2.1 × 150 mm, 1.7 μm) using a gradient consisting of water and acetonitrile with 10 mM ammonium formate. The MS analysis alternated between MS and data-dependent MS/MS scans using dynamic exclusion and the scan range was from 80–1000 m/z.

The samples destined for analysis by GC-MS were dried under vacuum for a minimum of 18 h prior to being derivatized under dried nitrogen using bistrimethyl-silyltrifluoroacetamide. Derivatized samples were separated on a 5% diphenyl/95% dimethyl polysiloxane fused silica column (20 m × 0.18 mm ID; 0.18 μm film thickness) with helium as carrier gas and a temperature ramp from 60° to 340 °C in a 17.5 minute period. Samples were analyzed on a Thermo-Finnigan Trace DSQ fast-scanning single-quadrupole mass spectrometer using electron impact ionization (EI) and operated at unit mass resolving power. The scan range was from 50–750 m/z.

Bioinformatics analysis

For the proteome, only peptides with TMT reporter ion signal intensities for all ten samples were used for further bioinformatics analysis. Any duplicate peptides were removed before the data was SumScale normalised. The two normalised data files were then merged together to give one 10vs10 file comparison. Any peptides which did not have intensity values in all twenty TMT reporter ion channels were filtered out and median values were taken of the control and treated samples respectively. A total of 1906 peptides were quantified across all liver samples.

For the metabolome, raw data was extracted, peak-identified and QC processed using Metabolon’s hardware and software. Raw data is available as Supplementary Table 6. Metabolites were identified by automated comparison of the ion features in the experimental samples against a reference library of more than 3000 purified standard compounds that included retention time/index (RI), mass to charge ratio (m/z), and chromatographic data (including MS/MS spectral data), and then curated by visual inspection for quality control using software developed at Metabolon48. Peaks were quantified using area-under-the-curve. A total of 673 metabolites were detected. The maximum percent missing data allowed was 20%. As a result, 602 metabolites were taken forward for bioanalytical analysis.

The language and statistical environment R49 together with the ade4 package50 method was employed in order to explore the relationship between the control and the treated samples. We regressed out the batch effects between TMT1 and TMT2 from the protein expression data, and to correct variation resulting from instrument inter-day tuning differences for the metabolome data, using the limma package removeBatchEffect51. For plotting of results, a Principal Component Analysis (PCA) was first performed. Missing data was imputed by the value of the median of the group for a given metabolite in order to perform the PCA analysis. A previous evaluation of different fold change (FC) rules have found that a 1.2-fold change could be regarded as indicative of a significantly varying protein in TMT-LC-MS/MS experiments52. Data used for the functional analysis were selected at the cut off values of q < 0.05 (p < 0.005) with FC > 1.2. Pairwise non-parametric Mann–Whitney U tests were performed and a p-value was attributed to each of the 1906 peptides and the 602 metabolites. The resulting p-values were adjusted by the Benjamini-Hochberg multi-test adjustment method for a high number of comparisons. Volcano plots were also constructed in order to visualize the differences in metabolite and protein expression for each of the comparisons. The aforementioned tests and plots were performed using in-house R scripts. The pathway analysis was done using the Thomson Reuters MetaCore Analytical Suite and/or the NIH Database for Annotation, Visualization and Integrated Discovery Bioinformatics Resources 6.7 (DAVID) using recommended analytical parameters53.