Methods

Cell culture

Hormone-independent MDA-MB-231 human breast cancer cells were maintained in 75 cm2 flasks (Corning, Tewksbury, USA) as previously described [18]. Cells were seeded at 106 cells in 75 cm2 flasks containing 10 mL Dulbecco’s Modified Eagle Medium (DMEM)-based maintenance medium. After a 24 hour (h) recovery period, cells were washed 3 times with 5 mL phosphate buffered saline (PBS), fresh medium added either with or without 100 mg/L glyphosate (Sigma-Aldrich Co Ltd, Gillingham, Dorset, UK), and culture continued for a further 6 days. Medium was refreshed at 24 and 96 h from the 1st day of treatment. The experiment was conducted in three biological replicates (3× negative controls and 3× glyphosate 100 mg/L, each condition in two technical replicates).

Sample preparation

Samples were lysed (PBS, 4% sodium dodecyl sulphate (SDS), 25 mM Tris(2-carboxyethyl)phosphine (TCEP), 1× complete ethylenediaminetetraacetic acid (EDTA)-free protease inhibitors (Roche Products Limited, Welwyn Garden City, UK) with 3 * 5 second (s) sonication on ice followed by heating at 95 °C for 10 minutes (min). Samples were alkylated with N-ethylmaleimide (50 mM) in the dark for 30 min at room temperature, centrifuged at 17,000g and the pellet discarded. Proteins were precipitated (with methanol–chloroform), dissolved in 100 µL of 0.1 M tetraethylammonium bromide (TEAB), 8 M urea, diluted 1:4 (urea to 2 M) and digested at 37 °C overnight with LysC (Wako Chemicals Europe, Neuss, Germany). Samples were further diluted 1:2.5 (urea 0.8 M) and digested at 37 °C for 16 h with trypsin (Thermo Fisher, Loughborough, UK). The digestion was stopped by adding trifluoroacetic acid (TFA) to a final concentration of 1%. Digested peptide samples were desalted using a tC18 SepPak plate (Waters UK, Elstree, UK), and 100 µg of each were labelled with Tandem Mass Tag (TMT)-6plex (Thermo Fisher). Labelled peptides were combined, dried, reconstituted in 1% TFA, desalted again as above, dried, and reconstituted in 5% formic acid.

Mass spectrometry analysis

The TMT labelled sample was analysed by RPLC-MS/MS/MS (145 min linear gradient) on a Fusion Tribrid Orbitrap operating in Data Dependent Acquisition mode (MultiNotch Simultaneous Precursor Selection method; MS1: profile mode, Orbitrap resolution 120 k, 400–1600 m/z, AGC target 400,000, 100 milliseconds (ms) maximum injection time, RF lens 60%; MS2: centroid mode, IonTrap, 10 dependent scans, 1.2 Th isolation window, charge states 2–6, 60 s dynamic exclusion, CID fragmentation (35%, activation Q 0.25), AGC target 10,000, 70 ms maximum injection time; MS3: profile mode, 5 precursors, 2 Th isolation window, Orbitrap resolution 30 k, 100–500 m/z, AGC target 50,000, 105 ms maximum injection time, HCD fragmentation (55%). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD013744.

Data analysis

The acquired raw file was searched with MaxQuant (1.6.0.13) against a human proteome Fasta database downloaded from UniProtKB. Because there was a single file to search, this allowed more variable modifications than normal without the search becoming impractically long. Variable modifications included in the search were “M-oxidation”, “N-terminal acetylation”, “deamidation (NQ)”, “Gln → pyroGlu”, “Phospho (STY)”, as well as two putative glyphosate-induced modifications: “A1” = glyoxylate-modified cysteine (+H 2 O 3 C 2 => expected monoisotopic mass shift +74.0003939305 Da) and “A2” = Glycine replaced by glyphosate (+H 3 O 3 CP => expected monoisotopic mass shift +93.9819804726 Da); finally, “N-ethylmaleimide” was unusually set as a variable modification (no fixed modifications) since it would compete with modification “A1” for the same sites. All false discovery rates (FDRs) were set to 1%. Dependent peptide search was ticked. Following MaxQuant analysis, data was reprocessed starting from the level of individual evidences for modified peptides (peptidoforms) using DC Biosciences’ TMT-labelled data processing scripts. Briefly, the Levenberg–Marquardt procedure was applied by column to normalise samples. Peptidoform reporter intensities were calculated as the sum of those of individual evidences and re-normalised as above. Peptide ratios were calculated (glyphosate vs average control), re-normalised as above, and summarized at protein groups level using an in house, mean based algorithm. Protein groups with a single identified peptidoform were excluded from the analysis. Moderated Welch’s t-tests were calculated and, in order to address the multiple hypothesis testing problem, p-value significance thresholds for 10, 20 and 30% False Discovery Rate were identified using the Benjamini–Hochberg procedure. Thresholds for significant ratios (fold change) were defined as the 5% upper and 5% lower tails of the ratios between individual controls and average control samples.

Results

In this study we set out to answer three distinct questions. First, are there any statistically robust global proteome changes in response to glyphosate treatment of mammalian cells? Second, can we identify modifications of cysteine residues as a result of the (putative) presence of glyoxylate, which is produced if glyphosate is metabolised (Fig. 1) [17]. Third, and most crucially, can we identify peptides where glyphosate has been directly incorporated in place of glycine?

Our results first confirmed our previous observation [18] that treatment of MDA-MB-231 cells with 100 mg/L glyphosate did not alter their growth characteristics (Additional file 1: Table S1). Statistical analysis of the ratios of global proteome changes between glyphosate treated and non-treated samples did not show significant changes (Fig. 2). Only two protein groups were found to be significantly up regulated; ADP/ATP translocase and serine/arginine-rich splicing factor 6. However, first these proteins are barely beyond the set thresholds and second, we would expect a small number of significant proteins under the null hypothesis with the criteria used.

Fig. 2 Volcano plot analysis of global proteomics changes after glyphosate treatment. X and Y axis: normalised log2 ratio and − log10 p-value of moderated Welch’s t-test. Vertical thresholds: upper and lower tails of the control-to-control ratios (5% most extreme log2 ratios in absolute value). Horizontal threshold: 30% False Discovery Rate (FDR) based on the Benjamini–Hochberg-procedure; there were no significant values at 10% and 20% FDR. Each dot represents a single protein group Full size image

We then tested samples for two different post-translational modifications (PTMs), which have been proposed to result from glyphosate exposure. Confident identification of new PTMs in proteomics is difficult, because peptide identification relies on matching rather than on full sequencing; peptide spectra are usually both hybrid and incomplete, so that spectra which can be fully de novo sequenced are rare. Proteomics peptide searches typically work at 1% FDR, which means that for any PTM, however unlikely to be truly present in the samples, it is to be expected that some peptides will nonetheless be identified. Normally, careful verification of putative PTMs is thus required, including analysis of synthetic peptides to show that their spectrum is similar to that of the identical, putative identifications. In this experiment, however, neither of the two putative PTMs of interest would be expected to be present in absence of glyphosate treatment. It was thus possible to use TMT labelling to identify and filter out any potential false discoveries. Indeed, by combining three treated and three untreated samples as one TMT-6plex labelled sample, we would expect a specific pattern (+/+/+/−/−/−) of reporter intensities for peptides bearing true glyphosate treatment induced-PTMs. By contrast, we would expect this pattern to only occur very rarely for peptides not bearing these PTMs: these would be putative peptides from proteins whose abundance would increase massively as a result of glyphosate treatment. However, as discussed above global analysis of the samples’ proteome failed to showcase any significant proteome changes as a response to treatment. Thus, in this experiment the pattern of peptides’ TMT reporter intensities constitutes a string filter to segregate real hits from false discoveries. Only glycine to glyphosate candidate peptides were identified in the search. As shown in Fig. 3, analysis of the TMT reporter intensity pattern of all candidates shows conclusively that they are all false discoveries, as none display the expected TMT pattern. True discoveries would be expected to have null or only trace reporter intensities in untreated channels (Fig. 3, red histogram bars), compared to a strong signal in treated channels (Fig. 3, blue histogram bars). Thus, we can confidently conclude that this analysis did not provide any evidence for the occurrence of either glyoxylation or substitution of glycine for glyphosate in proteins.

Fig. 3 Normalised TMT reporter intensities per TMT channel for all putatively identified glycine for glyphosate substituted peptides (indicated by “G(a2)” in the modified sequence). Since some such peptide discoveries would be expected under the null hypothesis (no substitution), we designed the experiment to use the isobaric pattern as a validation. Samples order is ∓ Glyphosate for replicate 1, then 2, then 3. True discoveries would be expected to have null or only trace reporter intensities in red (untreated) channels, compared to strong signal in blue (treated) channels. The data conclusively shows that all candidate substituted peptides are false discoveries Full size image

Discussion

We provide here a direct test using a proteomics approach of the assertion that glyphosate substitutes for glycine in proteins of mammals [10, 15]. Our results clearly show that glyphosate does not substitute for glycine in peptide chains (Fig. 3), which is in accord with previous observations in bacteria [16, 20]. In addition, our experiment allowed testing of glyphosate’s effects on the proteome profile of MDA-MB-231 cells and if the putative glyphosate metabolite glyoxylate could cause modifications of cysteine residues. No statistically significant effects were detected (Figs. 2, 3), which suggests that glyphosate does not have an effect on the proteome at the concentration tested.

In conclusion, our proteomics analysis proves the claim that glyphosate can substitute for glycine in proteins negatively affecting their structure and function is incorrect. Although our results will not come as a surprise to most of the scientific community, we believe they are nonetheless important in helping to clarify the debate on glyphosate toxicity in which many scientific hypotheses are considered as evidence of harm, ultimately influencing political debates, without being carefully tested in a controlled laboratory setting. We thus hope that our study will assist in focusing researchers’ attention on other aspects of glyphosate safety profiles, which remain to be investigated such as its impact on reproduction, development, carcinogenicity and microbiomes, especially of the gut [21,22,23].