Lipidomics and metabolomics communities comprise various informatics tools; however, software programs handling multimodal mass spectrometry (MS) data with structural annotations guided by the Lipidomics Standards Initiative are limited. Here, we provide MS-DIAL 5 for in-depth lipidome structural elucidation through electron-activated dissociation (EAD)-based tandem MS and determining their molecular localization through MS imaging (MSI) data using a species/tissue-specific lipidome database containing the predicted collision-cross section values. With the optimized EAD settings using 14 eV kinetic energy, the program correctly delineated lipid structures for 96.4% of authentic standards, among which 78.0% had the sn-, OH-, and/or C = C positions correctly assigned at concentrations exceeding 1 μM. We showcased our workflow by annotating the sn- and double-bond positions of eye-specific phosphatidylcholines containing very-long-chain polyunsaturated fatty acids (VLC-PUFAs), characterized as PC n-3-VLC-PUFA/FA. Using MSI data from the eye and n-3-VLC-PUFA-supplemented HeLa cells, we identified glycerol 3-phosphate acyltransferase as an enzyme candidate responsible for incorporating n-3 VLC-PUFAs into the sn1 position of phospholipids in mammalian cells, which was confirmed using EAD-MS/MS and recombinant proteins in a cell-free system. Therefore, the MS-DIAL 5 environment, combined with optimized MS data acquisition methods, facilitates a better understanding of lipid structures and their localization, offering insights into lipid biology.

Department of Applied Biological Science Tokyo University of Agriculture and Technology 3 5 8 Saiwai cho Fuchu Tokyo 183 8509 Japan

Department of Biotechnology and Life Science Tokyo University of Agriculture and Technology 2 24 16 Naka cho Koganei shi Tokyo 184 8588 Japan

Department of Medical Biochemistry Oslo University Hospital Sognsvannsveien 20 0372 Oslo Norway

Faculty of Medicine and Dentistry Palacký University Olomouc Hněvotínská 3 779 00 Olomouc Czech Republic

Graduate School of Medical Life Science Yokohama City University Yokohama Japan

Graduate School of Pharmaceutical Sciences Keio University Minato ku Tokyo 105 8512 Japan

Innovative Technology Laboratories AGC Inc 1 1 Suehiro cho Tsurumi ku Yokohama 230 0045 Japan

Institut de Pharmacologie Moléculaire et Cellulaire Université Côte d'Azur CNRS UMR7275 Inserm U1323 660 Route des Lucioles 06560 Valbonne France

Institute of Global Innovation Research Tokyo University of Agriculture and Technology 2 24 16 Naka cho Koganei shi Tokyo 184 8588 Japan

K K ABSciex Japan Shinagawa Tokyo 140 0001 Japan

Laboratory for Inherited Metabolic Disorders Department of Clinical Biochemistry University Hospital Olomouc Zdravotníků 248 7 779 00 Olomouc Czech Republic

RIKEN Center for Brain Science 2 1 Hirosawa Wako Saitama 351 0106 Japan

RIKEN Center for Integrative Medical Sciences 1 7 22 Suehiro cho Tsurumi ku Yokohama Kanagawa 230 0045 Japan

RIKEN Center for Sustainable Resource Science 1 7 22 Suehiro cho Tsurumi ku Yokohama Kanagawa 230 0045 Japan

Shimadzu Corporation 1 Nishinokyo Kuwabara cho Nakagyo ku Kyoto 604 8511 Japan

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