Bioorthogonal Click Chemistry in LC-MS lipidomics to trace lipid metabolism: from experimental data to high-throughput computational analysis
Metabolomics 2025. 22-26 June.
Stefano Bonciarelli1; Palina Nepachalovich2; Gabriele Lombardi Bendoula2; Jenny Desantis3; Michela Eleuteri3; Christoph Thiele4; Laura Goracci3; Maria Fedorova2
1Mass Analytica, Sant Cugat del Valles, Spain; 2Center of Membrane Biochemistry and Lipid Research, Faculty of Medicine Carl Gustav Carus, Technical University Dresden, Dresden, Germany; 3Department of Chemistry, Biology and Biotechnology, University of Perugia, Perugia, Italy; 4Life & Medical Sciences Institute, University of Bonn, Bonn, Germany
Abstract
Investigating lipid metabolism is crucial for understanding its role in health and disease. Recently, lipid metabolic tracing using bioorthogonal click chemistry has emerged as a powerful alternative to stable isotope tracers. [1,2] Thiele and colleagues showed that ω-alkyne-functionalized fatty acids, derivatized with azido-quaternary ammonium reporters, offer advantages like enhanced ionization and specific neutral loss, enabling high-throughput, sensitive lipid analysis. However, direct injection MS faces challenges, such as resolving isomeric species and possible occurrence of false positives from unaccounted in-source fragmentation. [3]
To address these limitations, we present an integrated LC-MS and bioinformatics platform for high-throughput lipid tracing using bioorthogonal click chemistry. [4] Synthetic standards and endogenously produced alkyne lipids were used to explore the LC-MS behavior of “clicked” lipid species with the C171 azido-quaternary ammonium reporter. Key factors like preferential adduct formation, in-source fragmentation (ISF), and MS/MS fragmentation patterns were annotated across 23 lipid subclasses. Fragmentation rules for each subclass were implemented in Lipostar2 software, enabling high-throughput annotation and quantification of 224,514 “clicked” lipids from 15 lipid classes.[4]
We validated the platform by tracing palmitic and oleic acids in HT1080 fibrosarcoma cells under fatty acid overload. High-throughput analysis identified 479 “clicked” lipids in palmitic acid-treated cells and 379 in oleic acid-treated cells, with species labeled singly, doubly, or triply. Distinct incorporation patterns were observed, including isomeric sphingolipid species, where the tracer incorporated into either the fatty acyl or sphingoid base. LC-MS revealed ISF products that would be indistinguishable from endogenous lipids by other methods. [4]
In conclusion, this integrated platform enables efficient, high-throughput analysis of “clicked” lipid tracers in LC-MS lipidomics.
[1] C. Thiele, C. Papan, D. Hoelper, K. Kusserow, A. Gaebler, M. Schoene, K. Piotrowitz, D. Lohmann, J. Spandl, A. Stevanovic, A. Shevchenko, L. Kuerschner, ACS Chem. Biol. 2012, 7, 2004–2011.
[2] C. Thiele, K. Wunderling, P. Leyendecker, Nat. Methods 2019, 16, 1123–1130.
[3] F.-F. Hsu, Anal Bioanal Chem 2018, 410, 6387–6409
[4] P. Nepachalovich, S. Bonciarelli, G. Lombardi Bendoula, J. Desantis, M. Eleuteri, C. Thiele, L. Goracci, M. Fedorova, Angew. Chem. Int. Ed. 2025, e202501884.
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