Untargeted UHPLC-HRMS Lipidomic Profiling of Ticks for Alpha-Gal Syndrome Research.

Lipids are a fundamental group of biomolecules that play diverse roles in biological systems, including energy storage, membrane architecture, and intra- and extracellular signaling. They are classified into eight major categories, and more than 50 000 distinct lipid structures are currently reported in the Lipid Maps Structure Database (LMSD). Given their importance in cellular metabolism and physiology, accurate identification and quantification of lipids are essential. In ticks, lipid metabolism is closely associated with physiological processes related to survival, pathogen transmission, and host interaction. Tick blood-feeding has also been linked to the development of alpha-gal syndrome (AGS), a delayed allergic reaction to mammalian-derived products, suggesting that characterization of tick lipid and glycolipid composition may provide insights into mechanisms of host sensitization. This chapter describes a protocol for untargeted lipidomic analysis of tick samples using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS). The workflow includes rapid sample freezing, tissue homogenization, solvent-based lipid extraction, protein precipitation, and sample reconstitution prior to analysis. Lipid separation is achieved using reverse-phase chromatography on a C18 column, followed by detection with a quadrupole time-of-flight (Q-TOF) mass spectrometer operating under optimized ionization conditions. The use of blanks and pooled quality control (QC) samples ensures analytical stability and reproducibility. The presented method enables comprehensive profiling of lipids in tick tissues and provides a reproducible platform for studies of tick biology, host-tick interactions, and tick-induced sensitization processes, supporting the mechanistic studies and the identification of potential lipid-based biomarkers.