A Comprehensive High-Resolution Targeted Workflow for the Deep Profiling of Sphingolipids.

Area of Science:

• Lipidomics
• Biochemistry
• Cellular Biology

Background:

• Sphingolipids are diverse biomolecules essential for cell membrane structure and function, including signaling and protein sorting.
• Accurate and comprehensive quantification methods are critical for a detailed understanding of the sphingolipidome.
• Current methods face challenges in resolving complex sphingolipid species.

Purpose of the Study:

• To develop and validate a targeted, high-resolution mass spectrometry-based workflow for accurate sphingolipid quantification.
• To improve the resolution of nearly isobaric and isobaric sphingolipid species.
• To establish a versatile tool for high-throughput sphingolipidomics.

Main Methods:

• Utilized a targeted reversed-phase liquid chromatography-high-resolution mass spectrometry (LC-MS) approach.
• Implemented an optimized extraction procedure and a segmented gradient for enhanced separation.
• Employed parallel reaction monitoring (PRM) with sphingolipid-specific fragmentation patterns for increased accuracy.

Main Results:

• Quantified 61 distinct sphingolipids in a RAW 264.7 cell line model system.
• Achieved quantification over a dynamic range of 7 orders of magnitude.
• Demonstrated low femtomole per milligram of protein detection limits, indicating high sensitivity.

Conclusions:

• The developed LC-MS workflow significantly enhances the accuracy and comprehensiveness of sphingolipid measurements.
• This method effectively resolves complex sphingolipid species, overcoming limitations of previous techniques.
• The workflow serves as a versatile and sensitive tool for high-throughput sphingolipidomics applications.