DISCOVERY LIPIDOMICS AND MAPPING OF EXOGENOUS FATTY ACID INCORPORATION INTO THE HELA LIPIDOME USING LC-IMS/MS AND LC-IMS/MS/MS

Importance of the topic

Exogenous fatty acid incorporation into cellular lipidomes provides critical insight into membrane dynamics, metabolic labeling techniques, and quantitative lipidomics. Using odd‐chain and fluorescent lipid analogs enhances the ability to trace lipid metabolism and distribution, supporting applications in cell biology, drug delivery, and disease biomarker discovery.

Objectives and study overview

This study aimed to profile how HeLa cells incorporate two exogenous lipid probes—heptadecanoic acid (C17:0) and a fluorescent BODIPY‐labeled palmitic acid analog (C16‐BODIPY)—into their endogenous lipidome. A combined discovery and targeted lipidomics approach, leveraging liquid chromatography coupled to ion mobility spectrometry and mass spectrometry (LC-IMS/MS and LC-IMS/MS/MS), was applied to detect, separate, and characterize modified phospholipids and sphingolipids.

Methodology and instrumentation

Cell Culture and Labeling

• HeLa cells were grown to 70% confluence and incubated with 50 µM heptadecanoic acid or C16-BODIPY for 24 hours.
• Labeled cells were harvested, snap-frozen in liquid nitrogen, and stored until extraction.

Sample Extraction and LC Conditions

• Cells were extracted by MTBE biphasic extraction, dried under nitrogen, and reconstituted in 60% water/40% acetonitrile with internal standard.
• Chromatography used an ACQUITY Premier CSH C18 column at 65 °C, with a binary gradient from 60% aqueous acetonitrile to 99% organic mobile phase.

Mass Spectrometry and IMS

• Analysis performed on a Waters SELECT SERIES Cyclic IMS with ESI in positive and negative modes, 60 000 resolution, and Leucine enkephalin lock mass.
• Ion mobility separations calibrated with Major Mix; single- and multipass HDMSE experiments applied collision energy ramps.

Used instrumentation

Waters ACQUITY Premier UPLC, ACQUITY Premier CSH C18 column (2.1 × 100 mm, 1.7 µm), Waters SELECT SERIES Cyclic Ion Mobility Spectrometer, Leucine enkephalin reference, nitrogen drift gas.

Main results and discussion

Fluorescent Lipid Incorporation

• C16-BODIPY was efficiently incorporated into phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) species, as visualized by fluorescence microscopy.
• IMS resolved BODIPY-labeled lipids from native lipids based on increased drift times and collisional cross sections.

Odd‐Chain Fatty Acid Labeling

• Heptadecanoic acid (C17:0) was rapidly incorporated and desaturated, generating a range of C17‐containing phospholipids.
• Multipass IMS enhanced separation of isobaric C17 species, enabling clear arrival time vs retention time maps.

Structural Characterization

• MS/MS fragmentation patterns confirmed headgroup identities and quantified the loss of HF from the BODIPY moiety.
• High-energy and low-energy spectra provided complementary insights into lipid backbone and probe structure.

Benefits and practical applications

• The combined LC-IMS/MS workflow allows comprehensive profiling of modified and native lipids in a single analysis.
• Fluorescent lipid analogs serve as robust internal standards for quantification and localization studies.
• Odd‐chain fatty acids act as non‐endogenous tracers for absolute quantification in lipidomics.

Future trends and opportunities

Further developments may include multiplexed fluorescent probes, integration with MS imaging, higher-throughput IMS technologies, and expansion to other cell types and organelles. Advances in ion mobility resolution and data processing algorithms will enhance structural elucidation and quantitation of lipid modifications.

Conclusion

This study demonstrates the power of LC-IMS/MS and cyclic IMS in mapping exogenous fatty acid incorporation into the HeLa lipidome. Fluorescent and odd‐chain lipid probes offer complementary advantages for tracing lipid metabolism and improving quantitative lipidomics workflows.

Reference

• Morstein J., Baker A. Discovery lipidomics and mapping of exogenous fatty acid incorporation into the HeLa lipidome using LC-IMS/MS and LC-IMS/MS/MS. bioRxiv. 2022. doi:10.1101/2022.02.14.480333v1
• Giles K., et al. Analytical Chemistry. 2019;91(13):8564-8573.