Iron is involved in many essential biological processes. Perturbed iron homeostasis, such as the accumulation of brain iron with age, can lead to oxidative stress and neuronal damage underlying neurodegenerative processes such as those observed in Alzheimer’s disease. Ferritin is the protein responsible for safe iron storage, and is conserved across taxa, including Caenorhabditis elegans. This microscopic nematode is a widely used animal model of ageing and is easily genetically manipulated. The C. elegans genome is well characterised, and importantly has homology with higher-order species providing an opportunity to study the relationship between neurodegeneration and iron metabolism.
Here we present an optimized protocol for purification and absolute quantitation of ferritin from cell lysate using custom (13C- and 15N-leucine/isoleucine) peptide standards, and parallel reaction monitoring (PRM)-based targeted mass spectrometry. Chromatographic separation was achieved within 60min (%RSD<2%), with 35% enrichment of ferritin signal from purified lysate. Linearity was established over 4 orders of magnitude (R2>0.99), with instrument limits of detection <1 fg on column. Inter-assay reproducibility was ~30%RSD. This method will be applied to an aged C. elegans populations to observe changes in iron homeostasis in ageing animals, before being transferred to other to other animal systems, such as the mouse. When conserved across taxa, these changes present a therapeutic target for age-related neurodegenerative diseases in humans.