LC-MS/MS-Based Untargeted Lipidomics Using MS-DIAL

Mass spectrometry (MS) has become the cornerstone of metabolomics and lipidomics because of its high sensitivity, specificity, and ability to analyze complex biological samples. However, the data complexity necessitates the use of robust computational tools. MS-DIAL is a versatile and user-friendly software for untargeted metabolomics and lipidomic workflows. It was launched as a universal program for untargeted metabolomics and supports multiple instruments (GC/MS, GC/MS/MS, LC/MS, and LC/MS/MS) and MS vendors (Agilent, Bruker, LECO, Sciex, Shimadzu, Thermo, and Waters). Common data formats such as netCDF (AIA) and mzML can also be managed. In addition, several MSP files, including EI and MS/MS spectra, were included as a "start-up kit." Moreover, MS-DIAL has an internal version of the Fiehn lab's GC/MS database (oriented by the FAME RI index), in silico retention time, and MS/MS database for LC/MS/MS-based lipidomics. Isotope-labeled tracking can be executed using an LC/MS project. This chapter provides two detailed protocols using LC-MS/MS for hydrophilic metabolome and lipidome analyses using MS-DIAL. Each protocol covers the key steps, including raw data import, peak detection, alignment, annotation, and export. Practical tips, troubleshooting strategies, and case studies are included to enhance the reproducibility and interpretation of the results.