Tiourea

Embryos from each of the experimental conditions (around 50 embryos per group) were collected at 48 hpf, deyolked, washed, frozen in liquid nitrogen and stored at −80 °C until further processing for the mass spectrometry experiment. Briefly, embryos were lysed with a buffer containing 7 M urea (Sigma, Burlington, MA, USA), 2 M tiourea (Sigma) and 50 mM ABC. Protein extracts were then quantified using a Bradford assay and 20 µg was utilized for the proteomic analysis. The reduction step was performed by adding dithiothreitol (DTT) (Sigma) to a final concentration of 10 mM and incubating at RT for 30 min. Subsequent alkylation with 30 mM (final concentration) iodoacetamide (IAA) (Sigma) was performed for 30 min in the dark at RT. Then, an additional reaction step was performed with 30 mM DTT (final concentration) for 30 min. This mixture was diluted to 0.9M urea using Miliq-water. The digestion step was performed using trypsin (Promega, Madison, WI, USA) at 1:50 w/w (enzyme/protein) and samples were incubated at 37 °C for 16 h. Digestion was quenched by acidification with acetic acid. The final step before mass spectrometry was the Vacuum Manifold platform. Then, samples were dried via a vacuum centrifuge and resuspended in 10 µL of 2% acetonitrile, 0.1% formic acid and 98% miliQ water.

MeHgCl, tiourea, 2-mercaptobenzimidazole (MBI), 2-mercaptobenzothiazole (MBT), acrylic acid (AA), ethylene glycol dimethacrylate (EGDMA) and azodiisobutyronitrile (AIBN) were purchased from Sigma-Aldrich (St. Louis, MO, USA); ethanol (EtOH) absolute (99.9%) was obtained from JT Baker (Radnor, PA, USA); MeHgCl standard solution 1000 mg L⁻¹ from Alfa Aesar (Tewksbury, MA, USA); and hydrochloric acid (HCl), sodium acetate (NaCH₃COO), acetic acid (CH₃COOH), sodium hydrogen phosphate (Na₂HPO₄), sodium dihydrogen phosphate (NaH₂PO₄), sodium hydroxide (NaOH), and sodium tetraborate (Na₂B₄O₇) were purchased from Merck (Darmstadt, Germany);. For the selectivity study, solutions were prepared from salt standards of lead nitrate, cadmium nitrate tetrahydrate, zinc sulfate monohydrate, and mercury dichloride (all acquired form Sigma-Aldrich). All the reagents used in this work were of analytical grade, and the solutions were prepared in ultrapure water (resistivity of 18.2 MΩ cm).
The samples were collected from the Batalha river (21.72 S, 49.20 W), and directly from the tap.
WARNING: MeHgCl appears as white microcrystals and is very toxic by inhalation, in contact with skin and if swallowed. Protection measures such as glasses, full face-shields, gloves and particulate respirators are required to manipulate it. Its disposal should also be carried out carefully.