Protocol for untargeted lipidomics of human serum using LC-TIMS-PASEF

Here, we present a protocol for untargeted lipidomics of human serum. We describe steps for project design, sample preparation, and reversed-phase liquid chromatography coupled with trapped ion mobility spectrometry and parallel accumulation serial fragmentation (LC-TIMS-PASEF). We then detail procedures for quality control and data processing. The protocol enables comprehensive lipid profiling from 5 μL of serum, yielding annotations across glycerophospholipids, glycerolipids, sphingolipids, sterols, and fatty acyls. The resulting datasets support statistical analysis and integration with complementary omics approaches.