PROTEOMICS AS A SENSITIVE TOOL TO INVESTIGATE IRON HOMEOSTASIS REGULATING MECHANISMS

Iron homeostasis is tightly regulated and altered iron levels are associated with anemia, organ damage by iron overload and iron-induced ferroptosis. Iron is mainly stored in hepatocytes and liver and spleen macrophages, where its release is regulated by the hormone hepcidin. Hepcidin expression is mainly regulated by binding of BMP ligands to their respective receptor complex on hepatocytes. Even though numerous proteins involved in the regulation of iron homeostasis have been identified and their function established, a system-wide molecular understanding is missing. We developed a mass spectrometry-based proteomics pipeline comprising optimized sample lysis, automated sample processing, mass spectrometry-based data-independent acquisition and user-friendly bioinformatic analysis to elucidate iron-induced changes on the whole proteome level of primary murine hepatocytes treated with FeNtA overnight and blood plasma from wildtype mice injected with iron-dextran (0.1g/kg) for 8 weeks. We reliably identified and quantified proteins involved in iron homeostasis in primary mouse hepatocytes such as BMP pathway components, including BMP receptors. In iron-treated hepatocytes our data showed an increase in ferritin and a reduction of serotransferrin and transferrin receptor 1, as well as an increase of ferroptosis-associated factors including Gluthatione S-transferase P1 (Gstp1). Interestingly, we also observed an increase in ferritin and Gstp1 and a reduction of serotransferrin/transferrin receptor 1 in the plasma of iron-injected wildtype mice. Additionally, other hepatic enzymes involved e.g. in energy metabolism, like Sorbitol dehydrogenase or Aldolase B, were increased in the plasma of these mice indicating iron-induced liver damage and thus could serve as novel markers of iron overloading conditions.