Prioritize biologically relevant ions for data-independent acquisition (BRI-DIA) in LC-MS/MS-based lipidomics...

Data-dependent acquisition (DDA) is the most commonly used MS/MS scan method for lipidomics analysis on orbitrap-based instrument. However, MS instrument associated software decide the top N precursors for fragmentation, resulting in stochasticity of precursor selection and compromised consistency and reproducibility. We introduce a novel workflow using biologically relevant lipids to construct inclusion list for data-independent acquisition (DIA), named as BRI-DIA workflow.

To ensure consistent coverage of biologically relevant lipids in LC-MS/MS-based lipidomics analysis.

Biologically relevant ion list was constructed based on LIPID MAPS and lipidome atlas in MS-DIAL 4. Lipids were extracted from mouse tissues and used to assess different MS/MS scan workflow (DDA, BRI-DIA, and hybrid mode) on LC-Orbitrap Exploris 480 mass spectrometer.

DDA resulted in more MS/MS events, but the total number of unique lipids identified by three methods (DDA, BRI-DIA, and hybrid MS/MS scan mode) is comparable (580 unique lipids across 44 lipid subclasses in mouse liver). Major cardiolipin molecular species were identified by data generated using BRI-DIA and hybrid methods and allowed calculation of cardiolipin compositions, while identification of the most abundant cardiolipin CL72:8 was missing in data generated using DDA method, leading to wrong calculation of cardiolipin composition.

The method of using inclusion list comprised of biologically relevant lipids in DIA MS/MS scan is as efficient as traditional DDA method in profiling lipids, but offers better consistency of lipid identification, compared to DDA method. This study was performed using Orbitrap Exploris 480, and we will further evaluate this workflow on other platforms, and if verified by future work, this biologically relevant ion fragmentation workflow could be routinely used in many studies to improve MS/MS identification capacities.