Targeted Detection of Phospholipid Aldehydes in Living Cells by Oxime Ligation

Abstract Oxidatively damaged lipids play critical roles in numerous human pathologies, yet methods to directly identify these species within living cells remain limited due to their transient and low‐abundance nature. Among lipid oxidation products, aldehyde‐containing lipids are significant due to their heightened reactivity and strong links to disease pathology. Here, we introduce OxiLox (oxime ligation to oxidized lipids), a method that leverages fluorescent hydroxylamine‐based probes to enable direct, chemoselective tagging of aldehyde‐containing lipids within living cells via rapid oxime ligation. Using confocal microscopy, we observe the subcellular localization of these oxidized lipids, highlighting prominent accumulation within perinuclear membranes, particularly under oxidative stress conditions induced by the ferroptosis activator RSL3. Coupled with high‐resolution mass spectrometry, our method identifies aldehyde lipid species, notably revealing the abundance of oxidized plasmenyl phosphatidylethanolamines in ferroptotic cells. We demonstrate that cellular labeling by hydroxylamine probes can be modulated by altering the expression of antioxidant enzymes such as aldose reductase (AKR1B1), underscoring the relationship between enzymatic antioxidant defenses and lipid aldehyde metabolism. Our work establishes OxiLox as a robust approach for direct, sensitive, and chemically precise detection of lipid aldehydes in live cells, offering new insights into the role of lipid oxidation in health and disease.