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Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry
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LC-MS and High-Throughput Data Processing Solutions for Lipid Metabolic Tracing Using Bioorthogonal Click Chemistry
Palina Nepachalovich1, Stefano Bonciarelli2, Gabriele Lombardi Bendoula1
1Center of Membrane Biochemistry and Lipid Research, Faculty of Medicine Carl Gustav Carus, Technical University Dresden, Tatzberg 47-49, Dresden, 01307, Germany.
Angewandte Chemie (International Ed. in English)
|April 24, 2025
Summary
This study introduces an advanced liquid chromatography-mass spectrometry (LC-MS) platform for tracing lipid metabolism in mammalian cells. The new workflow enhances accuracy and speed in analyzing complex lipid pathways and their bottlenecks.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Systems Biology
Background:
- Lipid metabolism tracing in mammalian cells is complex due to diverse lipid structures and pathways.
- Bioorthogonal click chemistry coupled with mass spectrometry (MS) has improved lipid analysis sensitivity and specificity.
- Existing methods face challenges in resolving complex lipidomes and high-throughput data processing.
Purpose of the Study:
- To develop an integrated analytical and bioinformatics platform for high-throughput lipid metabolism tracing.
- To enhance the capabilities of bioorthogonal click chemistry for lipid analysis using liquid chromatography (LC) coupled with MS.
- To enable detailed qualitative and quantitative analysis of lipidome fractions, including isomeric species.
Main Methods:
- Integration of liquid chromatography (LC) separation prior to mass spectrometry (MS) detection.
- Development of a software-assisted workflow (Lipostar2) for high-throughput data processing.
- Characterization of LC-MS behavior, including adduct formation and fragmentation, using synthetic and endogenous alkyne lipids.
- Derivation of specific fragmentation rules from tandem MS experiments for 23 lipid subclasses.
Main Results:
- LC separation successfully resolved labeled and unmodified lipids, enabling analysis of both fractions and isomeric species.
- Lipostar2 software, incorporating derived fragmentation rules, facilitated high-throughput annotation and quantification of labeled lipids.
- Metabolic pathways of palmitic and oleic acid alkynes were traced, revealing distinct incorporation patterns and identifying metabolic bottlenecks.
Conclusions:
- The developed integrated LC-MS platform provides a powerful tool for high-throughput tracing of lipid metabolism.
- This approach significantly advances the analytical capabilities for studying complex lipid transformations in mammalian cells.
- The platform enables deeper insights into metabolic pathways, lipid incorporation, and potential bottlenecks.
