The reliable acquisition of MS/MS data is a crucial step in lipidomics and metabolomics studies to identify analytes. This requires MS/MS fragmentation fast enough to generate high quality spectra over a sharp chromatographic peak. High sample throughput is a prerequisite for measuring large, statistically relevant sample cohorts such as those required in longitudinal clinical research studies. In this presentation, lipid extracts from the NIST reference SRM1950 plasma were used to investigate the number of lipids identified using three different reversed phase LC gradient times (6, 11, 20 minutes).
The MS data was acquired in ESI positive mode using a timsTOF Pro instrument (Bruker) in TIMS on and off modes. The acquisition of MS/MS data from the lipid extract was supported by the TIMS technology as it adds a complementary ion mobility dimension in addition to LC separation. This facilitated very fast MS/MS acquisition using a serial fragmentation of co-eluting precursors. The so-called Parallel Accumulation Serial Fragmentation (PASEF) mode allowed measuring MS/MS spectra at speeds up to 100 Hz. Resulting data were processed using the MetaboScape (Bruker) software considering all four dimensions: m/z, RT, intensity and CCS value. The open source LipidBlast MS/MS library (https://fiehnlab.ucdavis.edu/projects/LipidBlast) was used to assign lipid identities.
An identification of more than 200 lipids in 6 minute runs highlighted that PASEF MS/MS spectral quality was compatible with fast LC separations enabling up to 3x throughput compared to typical 20 minute LC runs. This demonstrates that complementary to an in-depth “ID as many as possible” approach, PASEF allows for a very fast lipid profiling based on MS/MS spectra. Additionally, we will show that even at reduced LC run times, the ion mobility separates co-eluting isobaric or isomeric compounds and provides accurate and reproducible CCS values for high confident lipid ID.