Critical Evaluation of Sphingolipids Detection by MALDI-MSI

Area of Science:

• Lipidomics
• Mass Spectrometry Imaging
• Biochemistry

Background:

• Sphingolipids play critical roles in cellular (patho)physiology, necessitating their visualization in tissues.
• Detecting sphingolipids via MALDI-MSI is challenging due to structural diversity, fragmentation, and varying abundance levels.
• Existing MALDI-MSI methods require optimization for robust sphingolipid analysis.

Purpose of the Study:

• To develop a robust pipeline for sphingolipid detection using MALDI-MSI.
• To establish best practices and define limitations for sphingolipid visualization via MALDI-MSI.
• To validate optimized protocols in cell cultures and a mouse breast cancer model.

Main Methods:

• Evaluated common MALDI-MS matrices (DHA, DHB in positive ion mode; DAN in negative ion mode) using sphingolipid reference standards.
• Assessed matrix performance for signal artifacts and fragmentation of complex sphingolipids.
• Validated optimized protocols in cell cultures by modulating sphingolipid levels and in PyMT mouse breast cancer tissue.

Main Results:

• Established optimal conditions for sphingomyelin (SM) and Sphingosine (Sph) detection using DHB in positive ion mode.
• Validated detection of ceramides and glycosphingolipids using DAN in negative ion mode.
• Documented extensive fragmentation of SM to S1P and C1P with DAN, causing artifactual C1P signals; confirmed lack of S1P/C1P detection with DHB positive ion mode.

Conclusions:

• Developed and validated a robust MALDI-MSI pipeline for sphingolipid visualization.
• Identified optimal matrix/ion mode combinations for specific sphingolipid classes, highlighting potential fragmentation artifacts.
• The study provides essential guidelines for accurate sphingolipid profiling in biological samples using MALDI-MSI.