Lc ms ultra chromasolv water

Protein identification was performed by mass spectrometry (MS) analysis [25 ]. Selected protein bands, found exclusively in samples containing NPs, were manually excised from the SDS-PAGE gel. Spots were dissected to smaller pieces and de-stained with 1:1 volume ratio of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate solution. Gel pieces were incubated in 200 mM NH₄HCO₃ solution for 20 min, washed two times with LC-MS Ultra CHROMASOLV^® water (Sigma-Aldrich, St. Luis, Missouri, USA), and dehydrated by covering with 100% acetonitrile. Reduction of proteins was performed with 10 mM dithiothreitol solution at 56°C for 45 min and alkylated by 55 mM iodoacetamide at room temperature for 30 min. Alkylation was stopped by addition of 25 mM NH₄HCO₃. Proteins were digested in gel with the MS grade modified trypsin (Sigma-Aldrich, Sigma-Aldrich, St. Luis, Missouri, USA) in 25 mM NH₄HCO₃ at 37°C overnight. Resulting peptides were extracted with 50% (v/v) acetonitrile / 5% (v/v) formic acid and concentrated in vacuum to 10 μL. Extracts were purified on StageTips C18 (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) according to the manufacturer's instructions and analysed using an electrospray ionization (ESI) ion trap-MS (MSD Trap XCT Plus, Agilent, USA) as described in Leonardi et al. [41 (link)].