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.

• MeSH
• data mining * metody   MeSH
• fosfatidylcholiny metabolismus   chemie   MeSH
• HeLa buňky MeSH
• hmotnostní spektrometrie metody   MeSH
• lidé MeSH
• lipidomika * metody   MeSH
• lipidy chemie   analýza   MeSH
• metabolomika metody   MeSH
• nenasycené mastné kyseliny metabolismus   chemie   MeSH
• software MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfatidylcholiny MeSH
• lipidy MeSH
• nenasycené mastné kyseliny MeSH

Liquid chromatography-mass spectrometry (LC-MS) is the key technique for analyzing complex lipids in biological samples. Various LC-MS modes are used for lipid separation, including different stationary phases, mobile-phase solvents, and modifiers. Quality control in lipidomics analysis is crucial to ensuring the generated data's reliability, reproducibility, and accuracy. While several quality control measures are commonly discussed, the impact of organic solvent quality during LC-MS analysis is often overlooked. Additionally, the annotation of complex lipids remains prone to biases, leading to potential misidentifications and incomplete characterization of lipid species. In this study, we investigate how LC-MS-grade isopropanol from different vendors may influence the quality of the mobile phase used in LC-MS-based untargeted lipidomic profiling of biological samples. Furthermore, we report the occurrence of an unusual, yet highly abundant, ethylamine adduct [M+46.0651]+ that may form for specific lipid subclasses during LC-MS analysis in positive electrospray ionization mode when acetonitrile is part of the mobile phase, potentially leading to lipid misidentification. These findings emphasize the importance of considering solvent quality in LC-MS analysis and highlight challenges in lipid annotation.

• Klíčová slova
• MS/MS annotation, adduct formation, lipidomics, lipids, liquid chromatography, mass spectrometry, metabolomics, method development, misidentification, solvent quality,
• Publikační typ
• časopisecké články MeSH