LIPIDOMIC PROFILING OF WILD TYPE AND FATTY ACID SYNTHASE (FASN) KNOCKOUT MICE WITH LC-cIMS-TOF MS AND MS/MS TECHNIQUES

Significance of the Topic

Fatty acid synthase (FASN) plays a central role in mammalian lipid metabolism by catalyzing the formation of short‐chain fatty acids from acetyl‐CoA. Disrupting FASN activity shifts acetyl‐CoA flux toward other anabolic and energy pathways, with important implications for understanding metabolic diseases, energy homeostasis, and the development of lipid‐targeted therapies.

Study Objectives and Overview

This work aimed to investigate how conditional knockout of the FASN gene in adult mice alters hepatic and cerebral lipid profiles. Using advanced lipidomic techniques, the study sought to:

• Compare lipid composition in wild‐type, heterozygous, and FASN knockout mice.
• Assess changes in triacylglycerols (TAGs), phospholipids, and other lipid classes.
• Evaluate the contribution of FASN activity to acetyl‐CoA‐derived lipid pools.

Methodology and Instrumentation

Animal Model and Sample Preparation:

• UBC‐Cre‐ERT2‐mediated conditional FASN knockout in eight‐week‐old mice via tamoxifen induction.
• Liver and brain tissues harvested five days post‐treatment and lipids extracted using the Folch protocol.

Liquid Chromatography and Mass Spectrometry:

• Reverse‐phase chromatography on an ACQUITY CSH C18 column with a gradient of acetonitrile/ammonium formate and isopropanol/acetonitrile buffers.
• Waters Select Series Cyclic IMS coupled to time‐of‐flight (TOF) MS with high‐ and low‐energy acquisition modes for both positive and negative electrospray ionization.
• Multipass high‐definition MS^E experiments to improve peak capacity and resolve isomeric lipids.

Main Results and Discussion

Alterations in TAG and Phospholipid Pools:

• Significant shifts in TAG species, notably C56:7, with corresponding changes in collisional cross section (CCS) and abundance ratios (ANOVA p < 0.005).
• Changes in phosphatidylglycerol species indicating remodeling of membrane lipid composition.

Multivariate Analysis:

• PCA demonstrated clear clustering among wild‐type, heterozygous, and knockout groups, underscoring distinct lipid signatures.

Enhanced Structural Resolution:

• Multipass IMS experiments increased separation of isomeric lipids, enabling confident assignment of fatty acyl chain composition.
• MS/MS fragmentation patterns confirmed the identity of key lipids via LipidBlast database matches.

Benefits and Practical Applications

The combined LC‐cIMS‐TOF MS/MS approach offers:

• High throughput and sensitivity for complex lipidome profiling.
• Improved isomer separation, critical for accurate biomarker discovery.
• Versatility for studies of metabolic regulation, drug effects, and disease biomarkers in preclinical and translational research.

Future Trends and Potential Applications

Emerging directions include:

• Integration of ion mobility data with machine learning for automated lipid annotation.
• Extended multipass separation and higher‐resolution IMS designs for deeper structural insight.
• Application to clinical specimens for diagnostic lipidomics and personalized medicine.

Conclusion

This study demonstrates that FASN knockout markedly alters lipid composition in mouse liver and brain, highlighting the enzyme’s impact on acetyl‐CoA distribution. The advanced LC‐cIMS‐TOF MS/MS platform provides a powerful tool for dissecting complex lipid networks and supports future investigations into metabolic regulation and therapeutic targeting.

Reference

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