Integrating Radical-Directed Dissociation and Isobaric Tagging for Simultaneous Lipid Structural Characterization and Quantification

Lipids play key biological roles in a variety of cellular functions, including signaling, energy storage, membrane structure, and cellular barriers. Mass spectrometry (MS) has emerged as a powerful lipidomic tool, offering high specificity and sensitivity. However, accurate quantification and structural characterization remain challenging due to lipid diversity and the prevalence of isomeric species that cannot be distinguished by MS alone. Conventional approaches relying on isotopically labeled standards are powerful but often costly and labor-intensive.

Free radical-initiated peptide sequencing (FRIPS) reagents, originally developed for protein analysis, generate radicals under tandem MS that drive radical-directed dissociation (RDD), yielding rich structural information. Recent work has applied FRIPS reagents with phosphatidylethanolamine lipids for isomer characterization, identifying their potential as a lipidomic strategy. Meanwhile, isobaric mass tags (IMTs) are a useful tool for relative quantification, but their application to lipids has required additional derivatization strategies. Building on our previous work functionalizing unsaturated lipids via aziridination to introduce a cyclic amine handle for tagging, we recognized that the design of FRIPS reagents shares critical features with IMTs.

In this work, we have developed a novel bifunctional tag that unifies RDD-based structural elucidation with isobaric quantification in a single reagent. This dual-function design enables simultaneous lipid isomer characterization and accurate relative quantification of unsaturated lipids across multiple lipid classes. By integrating aziridination chemistry with this new tag, we expand the scope of lipidomic analysis and demonstrate a streamlined strategy for addressing two central challenges in the field: structural resolution and quantification.