Using Data-Dependent and -Independent Hybrid Acquisitions for Fast Liquid Chromatography-Based Untargeted Lipidomics.

ABSTRACT

Untargeted lipidomics using liquid chromatography (LC) coupled with tandem mass spectrometry (MS) is essential for large cohort studies. Using a fast LC gradient of less than 10 min for the rapid screening of lipids decreases the annotation rate, because of the lower coverage of the MS/MS spectra caused by the narrow peak width. A systematic procedure is proposed in this study to achieve a high annotation rate in fast LC-based untargeted lipidomics by integrating data-dependent acquisition (DDA) and sequential window acquisition of all-theoretical mass spectrometry data-independent acquisition (SWATH-DIA) techniques using the updated MS-DIAL program. This strategy uses variable SWATH-DIA methods for quality control (QC) samples, which are a mixture of biological samples that were analyzed multiple times to correct the MS signal drift. In contrast, biological samples are analyzed using DDA to facilitate the structural elucidation of lipids using the pure spectrum to the maximum extent. The workflow is demonstrated using an 8.6 min LC gradient, where the QC samples are analyzed using five different SWATH-DIA methods. The use of both DDA and SWATH-DIA achieves a 1.7-fold annotation coverage from publicly available benchmark data obtained using a fast LC-DDA-MS technique and offers 95.3% lipid coverage, as compared to the benchmark data set from a 25 min LC gradient. This study demonstrates that harmonized improvements in analytical conditions and informatics tools provide a comprehensive lipidome in fast LC-based untargeted lipidomics, not only for large-scale studies but also for small-scale experiments, contributing to both clinical applications and basic biology.