Direct Mapping of Phospholipid Ferroptotic Death Signals in Cells and Tissues by Gas Cluster Ion Beam Secondary Ion Mass Spec-trometry (GCIB-SIMS).

Peroxidized phosphatidylethanolamine (PEox) species have been identified by Liquid Chromatography Mass Spectrometry (LC-MS) as predictive biomarkers of ferroptosis, a new program of regulated cell death. However, the presence and subcellular distribution of PEox in specific cell types and tissues have not been directly detected by imaging protocols. By applying Gas Cluster Ion Beam Secondary Ion Mass Spectrometry (GCIB-SIMS) imaging with a 70 keV (H 2 O) n + (n>28,000) cluster ion beam, we were able to map PEox with 1.2 micron spatial resolution at the single cell/subcellular level in ferroptotic H9c2 cardiomyocytes and cortical/hippocampal neurons after traumatic brain injury. Application of this protocol affords visualization of physiologically relevant levels of very low abundance (20 pmol/μmol lipid) peroxidized lipids in subcellular compartments and their accumulation in disease conditions.