Comparison of Data-Dependent Acquisition, Data-Independent Acquisition, and Parallel Reaction Monitoring in Trapped...

The parallel accumulation-serial fragmentation (PASEF) approach based on trapped ion mobility spectrometry (TIMS) enables mobility-resolved fragmentation and a higher number of fragments in the same time period compared to conventional MS/MS experiments. Furthermore, the ion mobility dimension offers novel approaches for fragmentation. Using parallel reaction monitoring (prm), the ion mobility dimension allows a more accurate selection of precursor windows, while using data-independent aquisition (dia) spectral quality is improved through ion-mobility filtering. Owing to favorable implementation in proteomics, the transferability of these PASEF modes to lipidomics is of great interest, especially as a result of the high complexity of analytes with similar fragments. However, these novel PASEF modes have not yet been thoroughly evaluated for lipidomics applications. Therefore, data-dependent acquisition (dda)-, dia-, and prm-PASEF were compared using hydrophilic interaction liquid chromatography (HILIC) for phospholipid class separation in human plasma samples. Results show that all three PASEF modes are generally suitable for usage in lipidomics. Although dia-PASEF achieves a high sensitivity in generating MS/MS spectra, the fragment-to-precursor assignment for lipids with both, similar retention time as well as ion mobility, was difficult in HILIC-MS/MS. Therefore, dda-PASEF is the method of choice to investigate unknown samples. However, the best data quality was achieved by prm-PASEF, owing to the focus on fragmentation of specified targets. The high selectivity and sensitivity in generating MS/MS spectra of prm-PASEF could be a potential alternative for targeted lipidomics, e.g., in clinical applications.