A comprehensive characterization of the lipidome from limited starting material remains very challenging. Here we report a high-sensitivity lipidomics workflow based on nanoflow liquid chromatography and trapped ion mobility spectrometry (TIMS). Taking advantage of parallel accumulation–serial fragmentation (PASEF), we fragment on average 15 precursors in each of 100 ms TIMS scans, while maintaining the full mobility resolution of co-eluting isomers. The acquisition speed of over 100 Hz allows us to obtain MS/MS spectra of the vast majority of isotope patterns. Analyzing 1 µL of human plasma, PASEF increases the number of identified lipids more than three times over standard TIMS-MS/MS, achieving attomole sensitivity. Building on high intra- and inter-laboratory precision and accuracy of TIMS collisional cross sections (CCS), we compile 1856 lipid CCS values from plasma, liver and cancer cells. Our study establishes PASEF in lipid analysis and paves the way for sensitive, ion mobility-enhanced lipidomics in four dimensions.

Milk lipids are known for a variety of biological functions, however; little is known about compositional variation across breeds, especially for Jaffarabadi buffalo, an indigenous Indian breed. Systematic profiling of extracted milk lipids was performed by mass spectrometry across summer and winter in Holstein Friesian cow and Jaffarabadi buffalo. Extensive MS/MS spectral analysis for the identification (ID) of probable lipid species using software followed by manual verification and grading of each assigned lipid species enabled ID based on (a) parent ion, (b) head group, and (c) partial/full acyl characteristic ions for comparative profiling of triacylglycerols between the breeds. Additionally, new triacylglycerol species with short-chain fatty acids were reported by manual interpretation of MS/MS spectra and comparison with curated repositories. Collectively, 1093 triacylglycerol species belonging to 141 unique sum compositions between the replicates of both the animal groups were identified. Relative quantitation at sum composition level followed by statistical analyses revealed changes in relative abundances of triacylglycerol species due to breed, season, and interaction effect of the two. Significant changes in triacylglycerols were observed between breeds (81%) and seasons (59%). When the interaction effect is statistically significant, a higher number of triacylglycerols species in Jaffarabadi has lesser seasonal variation than Holstein Friesian.