Electron-activated dissociation for structural resolution of lipid isomers: Principles, performance, and the path to...

Lipids encode critical biological information not only through their class and sum composition, but also through fine structural features, including fatty acyl chain stereospecific numbering (sn) position in glycerolipids and glycerophospholipids, and carbon-carbon double-bond (C=C) location and geometry. These isomeric features are vital for modulating membrane organization and cell signaling and have been correlated with numerous disease phenotypes. However, conventional high-throughput lipidomics workflows, typically relying on collision-induced dissociation (CID), are often unable to resolve this isomer-level detail. Electron-activated dissociation (EAD) is an umbrella term for a family of electron-based fragmentation methods. Within this family, the low-energy regime, historically known as electron impact excitation of ions from organics (EIEIO), uses radical chemistry to induce precise cleavages along the lipid backbone and acyl chains. This mechanism generates direct, diagnostic fragment ions that enable the comprehensive resolution of sn- and C=C position isomers, branching, hydroxylation and, in selected cases, cis/trans geometry from intact complex lipids. Although EAD's powerful capability for structural elucidation has been demonstrated, its community-wide implementation in routine lipidomics remains an emerging transition. This systematic review thoroughly details EAD fragmentation principles across major lipid classes, including triacylglycerols, glycerophospholipids, sphingolipids, and cardiolipins, and surveys the current literature for its structural applications. Consequently, I evaluate the critical bottlenecks to broad adoption and discuss how recent advancements in instrument sensitivity, data-independent acquisition (DIA) workflows, and automated EAD-aware bioinformatics software are effectively addressing these challenges. Ultimately, I outline the necessary steps to fully integrate EAD into routine, high-throughput standard practice for isomer-resolved lipidomics.