Identifying Double-Bond-Positions of Phospholipids in Mouse Liver by Using Simultaneous Positive/Negative Ion Switching Analysis of LCMS-9050 and OAD-MS/MS

Significance of the Topic

Lipids are critical for cell function and biomarker discovery, but locating double bonds within fatty acyl chains remains analytically challenging. Advanced structural lipidomics techniques enable detailed insight into lipid metabolism in health and disease.

Objectives and Study Overview

This study aimed to develop a robust workflow for pinpointing double-bond positions in phosphatidylcholine species extracted from mouse liver. Combining oxygen attachment dissociation (OAD) and collision-induced dissociation (CID) with simultaneous positive/negative ion switching on a high-resolution LC-MS platform, researchers sought to identify isomeric phospholipids accurately.

Methodology and Instrumentation

• Sample preparation: Mouse liver homogenized in methanol, followed by chloroform extraction, aqueous partitioning, and centrifugation.
• Liquid chromatography: Shimadzu Nexera X3 system with C18 column, gradient elution using 20 mM ammonium formate (A) and ACN/IPA (B), flow rate 0.3 mL/min.
• Mass spectrometry: Shimadzu LCMS-9050 Q-TOF operated in DDA mode with rapid positive/negative ion switching, collision energies of 25 V (positive) and 35 V (negative).
• OAD source: Microwave-driven O/OH radical generation introduced into the collision cell for oxygen attachment dissociation.
• CID: Used to determine phospholipid headgroup and fatty acid composition.

Main Results and Discussion

• Detection of PC(36:4) isomers at m/z 782.5695 ([M+H]+) and 826.5604 ([M+HCOO]−).
• CID-based spectra revealed overall chain length (36 carbons) and four double bonds but could not resolve positional isomers.
• OAD-MS/MS fragmentation generated diagnostic neutral losses corresponding to specific double-bond cleavage sites in fatty acyl chains.
• Two distinct peaks (A and B) were characterized: Peak A as 18:2_18:2 and Peak B as 16:0_20:4 compositions, with precise localization of double bonds within each acyl chain.

Benefits and Practical Applications

• Enables unambiguous assignment of double-bond positions in complex lipid extracts.
• Enhances structural lipidomics workflows for biomarker discovery and metabolic research.
• Applicable to quality control and detailed profiling in pharmaceutical and food industries.

Future Trends and Applications

• Integration of OAD-MS/MS with automated data analysis and lipid databases for high-throughput screening.
• Expansion of the approach to other lipid classes (e.g., phosphatidylethanolamines, sphingolipids).
• Combination with ion mobility and advanced fragmentation strategies for deeper structural elucidation.
• Application in clinical lipidomics for disease diagnostics and personalized medicine.

Conclusion

The study demonstrates that coupling OAD with simultaneous positive/negative ion switching on a high-resolution LC-MS platform provides reliable localization of double bonds in phosphatidylcholines. This approach addresses a key limitation in lipid structural analysis, offering detailed insights into lipid metabolism.

Reference

1. Takahashi H. et al. Mass Spectrometry. 2019, S0080.
2. Uchino H. et al. Commun Chem. 5, 162 (2022).