Icp ms is mix1 1

ICP-MS analysis of metal levels was performed as reported previously [24] (link). Cell pellets collected for metal analysis by ICP-MS were re-suspended in 50 µL of concentrated nitric acid (Aristar, BDH, Kilsyth, VIC, Australia) and left to digest overnight. Samples were heated for 20 min at 90°C to complete the digestion. The volume of each sample was reduced to approximately 40–50 µL after digestion, then 1 mL of 1% (v/v) nitric acid diluent was added to each cell sample. Measurements were made using an Agilent ICPMS 7700x series ICP-MS instrument under operating conditions suitable for routine multi-element analysis. The instrument was calibrated using 0, 5, 10, 50, 100 and 500 parts per billion (ppb) of certified multi-element ICP-MS standard calibration solutions (ICP-MS-CA12-1, ICP-MS-CAL-3 and ICP-MS-CAL-4, Accustandard) prepared in 1% (v/v) nitric acid for Cu, Fe, Zn and Mn. A certified internal standard solution containing 200 ppb of Yttrium (Y⁸⁹) via T-piece was used as an internal control (ICP-MS- IS-MIX1-1, Accustandard). Results were expressed as micromole per liter concentrations of metal (µmol/L). The concentrations of Cu and other metals were calculated as µg of metal per mg of protein based on the protein concentration of the aliquot taken from each sample.

Tissue samples were coded and processed for inductively coupled mass spectroscopy analysis at the Florey Institute of Neuroscience and Mental Health. Tissue samples were lyophilised and then digested with nitric acid (65% Suprapur, Merck) overnight, followed by heating at 90°C for 20 min using a heat block. Samples were then removed from the heat block and an equivalent volume of hydrogen peroxide (30% Aristar, BDH) added to each sample. Once samples had finished digesting, they were heated for a further 15 mins at 70°C. Samples were then diluted with 1% nitric acid diluent. Measurements were made using an Agilent 7700 series ICPMS instrument under routine multi-element operating conditions using a Helium Reaction Gas Cell. The instrument was calibrated using 0, 5, 10, 50, 100 and 500 ppb of certified multi-element ICPMS standard calibration solutions (ICP-MS-CAL2-1, ICP-MS-CAL-3 and ICP-MS-CAL-4, Accustandard) for a range of elements, and we also utilised a certified internal standard solution containing 200 ppb of Yttrium (Y89) as a control (ICP-MS-IS-MIX1-1, Accustandard).

Frozen cell pellets were lysed in unbuffered 0.5% SDS and cleared by ultracentrifugation. Protein concentration in the lysates was measured with BCA and 500ug of each sample was diluted to a final volume of 400uL with additional 0.5% SDS and freeze dried. Aliquots of 400uL 0.5% SDS as blank controls were included as background controls. The samples were assayed by inductively coupled plasma mass spectrometry (ICPMS; Agilent 7700) under routine multi-element operating conditions using a Helium Reaction Gas Cell, according to a previously published method [34 (link)]. Briefly, samples were treated with concentrated Nitric Acid (65%, Suprapur, Merck) and digested for six hours at room temperature, then heated at 90°C for 20 min to complete the digestion. The instrument was calibrated using 0, 5, 10, 50, 100 and 500 ppb of certified multi- element ICPMS standard calibration solutions (ICP-MS-CAL2-1, ICP-MS-CAL-3 and ICP-MS-CAL-4, Accustandard) for a range of elements, and a certified internal standard solution containing 200 ppb of Yttrium (Y89) as an internal control (ICP-MS-IS-MIX1-1, Accustandard). Cellular copper levels are presented as a ratio of cellular magnesium content.

Inductively coupled plasma mass spectrometry (ICP-MS) was used to measure bulk metal concentration in tissue samples as described elsewhere (Maynard et al., 2002 (link)). Briefly, perfused brain and spinal cord tissue samples were lyophilized then digested in HNO₃ (65% Suprapur, Merck) overnight. Liver and quadriceps muscles were used as control non-CNS tissue. Tissues were heated at 90^oC before the addition of H₂O₂ (30% Aristar, BDH). Samples were left to stand for ~30 min, before further heating at 70^oC. The average reduced volume was determined and samples were further diluted with 1% HNO₃. Measurements were made using an Agilent 7700 series ICP-MS instrument using a Helium Reaction Gas Cell and 200 ppb of Yttrium (Y89) as an internal control (ICP-MS-IS-MIX1-1, Accustandard). Results are expressed as micrograms of metal per gram of wet weight tissue (μg/g).

Cell pellets were digested in a minimal volume of concentrated nitric acid (Suprapur, Merck, Australia) overnight, followed by heating of the samples at 90 °C for 20 min to complete the digestion. The reduced volume after digestion (~20 μL) was diluted up to a final volume of 1 mL in Milli-Q water. Measurements of Se, Cu, Zn, Fe were made using an Agilent 8900 triple quadrupole inductively coupled plasma mass spectrometer (Agilent Technologies, Mulgrave, Australia) under operating conditions previously described [72 (link)]. The instrument was calibrated using blank, 10, 50, and 100 parts per billion (ppb) of a certified multi-element ICPMS standard solution (ICP-MS- CAl2–1, Accustandard, CT, USA). A certified internal standard solution containing 100 ppb of Yttrium via a T-piece was used as an internal control (ICP-MS- IS-MIX1-1, Accustandard, CT, USA). PBS and sample preparation blanks (containing Milli-Q water and nitric acid) were measured in each ICP-MS experiment to monitor for metal contamination. Data are expressed as μmol element/mg protein.

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).

Sub-confluent cultures of TBCP-1 cells (5 replicates/condition) were treated for 72 h with vehicle alone (DMSO control) or neratinib (300 nM and 500 nM) and the cells pelleted by centrifugation. Fifty microlitres of concentrated nitric acid (65% v/v, Suprapur, Merck) was added to each cell pellet overnight at room temperature. Samples were heated at 90 °C for 20 min, and final volumes made up to 500 μl 1% (v/v) nitric acid. Iron content was measured using an Agilent 7700 series ICP-MS instrument under routine multi-element operating conditions in a Helium Reaction Gas Cell. The instrument was calibrated using 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) for a range of elements. A certified standard solution containing 200 ppb of yttrium (Y89) was used as an internal control (ICP-MS-IS-MIX1-1, Accustandard). The raw ppb results were normalised to final volume and converted to ng/10⁶ cell of metal using the formula: Final concentration (ng/10⁶ cell) = Raw ppb value (ng/mL) × sample volume (0.5 ml)/number of cells × 10⁶.