Tissues were homogenized and sonicated in protein extracting solution (50 mM SDS, 10 mM NaCl, 50 mM Tris-HCL, pH 7.4), filtered and injected to a 1200 HPLC system composed of a thermostated autosampler set to 4°C, a vacuum membrane degasser, a binary pump, a column oven compartment, and a UV-Vis diode array detector (Agilent). The size exclusion chromatography (SEC) column was a TSK gel 3000SW 7.8 × 30 mm, 10 μm particle size with 50 mM NH4Ac buffer in 0.1% MeOH at pH 7.4 at the mobile phase at 0.5 mL min–1 (Tosoh Global). The outlet of the HPLC system was connected to the ICP-MS-MS nebulizer by a 65 cm PEEK capillary of 0.17 mm of internal diameter. The ICP-MS was operated in time-resolved analysis with an integration time of 0.15 seconds per isotope. A 5-point calibration was generated for 75As, 63Cu, 111Cd, 56Fe, 60Ni, 78Se, 207Pb, 34S, 31P, 55Mn, 59Co and 66Zn with a spiked FBS sample in order to avoid column contamination by free metals and to replicate the protein matrix of the real samples. FBS was spiked with 0.2 ppm of Pb, Cd, Se, Co, Mn, and P with inorganic salts and incubated for 1 hour at 37°C. The unbound metals were filtered out and a total metal analysis was carried out in the remaining FBS. An aliquot of metal-enriched protein sample was filtered and diluted 1:1 with the SEC mobile phase and injected in increasing volumes to generate a calibration curve.

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