Mapping sterols in a marine flatworm-algae-system using MALDI-2 TIMS MS-imaging

Importance of the topic

Photosymbiotic partnerships between marine hosts and dinoflagellate algae are key to nutrient exchange in ocean ecosystems. In many of these associations, sterols produced by symbionts are transferred to hosts that cannot synthesize them. Understanding the spatial distribution of these sterols sheds light on host metabolism, symbiont contribution and broader ecological function.

Objectives and Overview

This study aimed to visualize and differentiate over 30 sterol species in the acoel flatworm Waminoa sp. and its endosymbiotic algae Amphidinium and Symbiodiniaceae. By combining laser post-ionization MALDI-2 with trapped ion mobility spectrometry (TIMS) on a timsTOF fleX instrument, the work seeks to achieve high-resolution mass spectrometry imaging to map sterol exchange and localization within host tissues.

Methodology

Waminoa individuals hosting dinoflagellates were fixed in a hydroxyethylcellulose/gelatin matrix and cryosectioned at 14 µm thickness onto ITO slides. A DHAP matrix was sublimated under controlled vacuum and temperature to ensure uniform coating. Sections were analyzed in positive ion mode targeting m/z 250–655 with and without TIMS separation. A 5 µm pixel size was used to approach cellular resolution. Ion images were processed with SCiLS Lab and TDF viewer software.

Instrumental Setup

• Bruker timsTOF fleX mass spectrometer modified for MALDI-2 post-ionization at 266 nm, 1 kHz repetition rate
• Trapped ion mobility spectrometry (TIMS) ramp from 0.6 to 1.6 1/k0 with 659 ms scan time
• Mass range m/z 300–1000 (without TIMS) and 250–655 (with TIMS)
• Home-built sublimation apparatus for DHAP deposition at 130 °C and 3 mbar

Main Results and Discussion

MALDI-2 enhanced sterol ion yields by up to 2–3 orders of magnitude, enabling detection of 32 distinct sterol species as [M–H2O+H]+ and radical [M–H2O]•+ ions. TIMS provided an extra separation dimension, yielding characteristic broadened mobility peaks that helped distinguish sterols from matrix noise. Key observations include:

• Cholesterol exhibited a nearly uniform tissue distribution.
• Stigmasterol showed pronounced accumulation in the syncytial gut region, suggesting localized uptake or storage.
• Saringosterol patterns resembled cholesterol centrally, with depletion at the periphery.
• Host phospholipid signals outlined flatworm morphology, while chlorophyll a fragments and unknown ions localized to individual algal cells (~9–13 µm diameter).

Benefits and Practical Applications

The combined MALDI-2/TIMS imaging workflow delivers cellular-level mapping of neutral sterols without chemical derivatization. Enhanced sensitivity and selectivity enable studies of sterol metabolism in symbiotic systems, quality control of sterol content in aquaculture, and targeted analysis in biomedical and environmental research.

Future Trends and Potential Applications

• Application to other host-symbiont systems, corals and sponges
• Integration with complementary imaging modalities (e.g., fluorescence microscopy) for correlative analysis
• Development of automated TIMS peak deconvolution for high-throughput sterol annotation
• Extension to other low-abundance neutral lipids and hormone precursors in tissue imaging

Conclusion

This work establishes MALDI-2 TIMS MS imaging as a powerful approach for sensitive, high-resolution mapping of sterols in marine symbioses. The distinct spatial patterns of cholesterol, stigmasterol and other phytosterols in Waminoa sp. highlight sterol species-specific roles beyond energy supply and reveal conserved lipid transfer mechanisms in dinoflagellate symbioses.

References

• Soltwisch L et al. Science 348:211–215 (2015)
• Soltwisch L et al. Analytical Chemistry 92:8697–8703 (2020)
• Bien T et al. Analytical and Bioanalytical Chemistry, in press (2020)