4D-Lipidomics™ workflow for increased throughput

Importance of 4D-Lipidomics

Lipids are key components of cell membranes, energy storage and signaling pathways. Their structural diversity and wide concentration range create analytical challenges. High confidence in lipid annotation and rapid analysis are crucial for applications in clinical research, large cohort phenomics and QA/QC workflows.

Objectives and Study Overview

This study demonstrates a 4D-Lipidomics workflow combining trapped ion mobility, high-speed PASEF acquisition and CCS-aware data processing. Using NIST SRM 1950 reference plasma extracts, it evaluates throughput, annotation confidence and reproducibility across LC runtimes of 5, 10 and 20 minutes.

Methods and Instrumentation

A methyl-tert-butyl ether extraction protocol yielded lipid extracts analyzed by RP-UHPLC and timsTOF Pro in PASEF MS/MS mode. Key parameters included:

• UHPLC: Triart C18 column (100×2.1 mm, 1.9 μm), gradients of 5, 10 and 20 min
• Mobile phases: A – MeCN/H2O 60:40, B – IPA/MeCN/H2O 90:8:2, both with 10 mM ammonium formate and 0.1% formic acid
• ESI source in positive and negative mode, PASEF ramps of 100 ms
• Internal calibration: sodium formate; mobility calibration: Agilent Tunemix
• Data processing: MetaboScape 2021b with T-ReX 4D, rule-based lipid annotation and optional CCS matching via LipidBlast library

Main Results and Discussion

• Single-injection PASEF achieved >65 % MS/MS coverage across m/z and retention time without replicates
• Feature extraction and filtering (CV <20 % across five injections) yielded robust datasets for annotation
• Rule-based annotation supported >40 lipid subclasses; complementary LipidBlast CCS library increased coverage and confidence
• 4D Kendrick Mass Defect plots highlighted homologous series and flagged annotation outliers
• At 20 min gradient, 362 unique lipids were identified; even at 5 min, 271 lipids (75 %) were annotated with high confidence
• Measured CCS values showed excellent correlation (R2 = 0.9987) between 5 and 20 min runs, demonstrating chromatography-independent CCS reliability

Benefits and Practical Applications

The workflow delivers high throughput lipid profiling with confident annotation from single injections. CCS-aware rule-based and library-based approaches reduce false positives and support spatial distribution studies via SpatialOMx coupling to MALDI imaging. This makes it suitable for large-scale clinical and industrial projects requiring fast turnaround.

Future Trends and Potential Applications

Integration of CCS-driven annotation with imaging mass spectrometry will enable spatial lipidomics in tissues. Further expansion of CCS libraries and advanced machine learning for de novo lipid identification can deepen biological insights. High-throughput 4D workflows are poised to accelerate biomarker discovery and precision medicine studies.

Conclusion

The 4D-Lipidomics workflow on timsTOF Pro/fleX offers rapid, high-confidence lipid profiling. PASEF-enabled MS/MS coverage, T-ReX 4D processing, rule-based annotation and CCS matching ensure reliable results even at runtimes as short as 5 min, supporting large cohort and spatial lipidomics applications.

Reference

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