Agilent 7700 series icp ms instrument

To measure pH-dependent release of iron from iron-binding proteins, 1-ml aliquots of 0.5 mg ml⁻¹ holo-hemoglobin, ferritin, and transferrin were dissolved in ACCM-2 inorganic basal buffer (26 (link)) without FeSO₄ at either pH 7.3 or 4.75. These solutions were incubated for 2 h at 37°C before being passed through a 3-kDa filter (Amicon Ultra-0.5 centrifugal filter unit; MilliporeSigma, Burlington, MA) to separate iron bound to intact proteins from the free iron. To process, 0.1 ml concentrated HNO₃ was added to the filtrates to extract free iron overnight at room temperature, heated to 80°C for 1 h, and then diluted to a final volume of 5 ml with double-distilled water (ddH₂O). Iron content was measured by inductively coupled plasma mass spectroscopy (ICP-MS) using an Agilent 7700 series ICP-MS instrument (Agilent Technologies, Santa Clara, CA).

ICP-MS was performed as previously described [56 (link)]. Briefly, ipsilateral and contralateral cortical brain homogenates were lyophilized and the dry material was digested with 50 µL of Nitric Acid (HNO₃, 65% Suprapur, Merck) overnight. The samples were further digested by heating to 90^°C for 20 min using a heating block. Samples were removed from the heating block and an equivalent volume of 50 µL of Hydrogen Peroxide (H₂O₂, 30% Aristar, BDH) was added to each sample. Samples were allowed to stop digesting for ~ 30 min before heating again for 15 min at 70^°C. The average reduced volume was determined and the samples further diluted with 1% HNO₃. All measurements were performed on an Agilent 7700 Series ICP-MS instrument (Agilent Technologies, CA, USA) under routine multi-element operating conditions using a Helium Reaction Gas Cell. The instrument was calibrated with 0, 5, 10, 50, 100, and 500 ppb of certified multi-element ICP-MS standard calibration solutions (ICP-MS-CAL2-1, ICP-MS-CAL-3 and ICP-MS-CAL-4, Accustandard, CT, USA) for a range of elements. A certified internal standard solution containing 200 ppb of Yttrium (Y89) was used as the internal control (ICP-MS-IS-MIX1-1, Accustandard). Metal content (µg/g) was normalised to protein concentration which was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific).