Molecular insights into symbiosis—mapping sterols in a marine flatworm-algae-system using high spatial resolution MALDI-2-MS imaging with ion mobility separation

Abstract

Waminoa sp. acoel flatworms hosting Symbiodiniaceae and the related Amphidinium dinoflagellate algae are an interesting model system for symbiosis in marine environments. While the host provides a microhabitat and safety, the algae power the system by photosynthesis and supply the worm with nutrients. Among these nutrients are sterols, including cholesterol and numerous phytosterols. While it is widely accepted that these compounds are produced by the symbiotic dinoflagellates, their transfer to and fate within the sterol-auxotrophic Waminoa worm host as well as their role in its metabolism are unknown. Here we used matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging combined with laser-induced post-ionization and trapped ion mobility spectrometry (MALDI-2-TIMS-MSI) to map the spatial distribution of over 30 different sterol species in sections of the symbiotic system. The use of laser post-ionization crucially increased ion yields and allowed the recording of images with a pixel size of 5 μm. Trapped ion mobility spectrometry (TIMS) helped with the tentative assignment of over 30 sterol species. Correlation with anatomical features of the worm, revealed by host-derived phospholipid signals, and the location of the dinoflagellates, revealed by chlorophyll a signal, disclosed peculiar differences in the distribution of different sterol species (e.g. of cholesterol versus stigmasterol) within the receiving host. These findings point to sterol species-specific roles in the metabolism of Waminoa beyond a mere source of energy. They also underline the value of the MALDI-2-TIMS-MSI method to future research in the spatially resolved analysis of sterols.

De novo phytosterol synthesis in animals

“…Matrix application and data acquisition. The matrix 2′,5′-dihydroxyacetophenone (DHAP; Merck) was applied by sublimation and the data were acquired on a modified timsTOF fleX instrument (Bruker Daltonics, Bremen, Germany) (Soltwisch et al, 2020) as described in (Bien et al, 2021). The mass resolving power of this hybrid QTOF-type instrument is about 40,000 (fwhm) in the investigated m/z range of 300-1500.…”

Spatial distribution of isobaric androgens in target tissues using chemical derivatization and MALDI-2 on a trapped ion mobility quadrupole time-of-flight instrument

“…Laser-induced post-ionization was achieved using a diode-pumped, actively Q-switched, frequency-quadrupled Nd:YAG laser operated at 1 kHz (NL 204-1k-FH, EKS-PLA, Vilnius, Lithuania) as described previously. 12 The instrument was operated in positive ion mode, and ions between m / z 300 and 800 were accumulated from 769 laser shots per 50 μm pixel. For TIMS analysis, a ramp time of 769 ms was chosen including a range for 1/ K 0 from 1.1 to 1.75 Vs cm −2 .…”

“…10 To overcome these issues, we here assessed, for the rst time, the use of laser-induced post-ionization (MALDI-2), combined with trapped ion mobility separation (TIMS) (MALDI-2-TIMS) in the identication and distribution analysis of isobaric derivatized androgen in a murine tumour xenogra model. MALDI-2 is a recently introduced method for enhanced MSI of numerous classes of biomolecules including sterols, 12 in tissue sections at high lateral resolution. 13 In this proof of concept study, two derivatization reagents, namely Girard-T (GT) and Dansyl Hydrazine (DS) were evaluated using a timsTOF eX MALDI-2 platform from Bruker.…”

Advances in MALDI Mass Spectrometry Imaging Single Cell and Tissues

“…Critical parameters for initiation of the secondary ionization processes are pressure of the cooling gas in the ion source, laser wavelength, pulse energy, and delay between the two laser pulses. The technology could enable sensitive MALDI-MS imaging with a lateral resolution in the low micrometer range (Soltwisch et al, 2015;Soltwisch et al, 2020;Bien et al, 2021b).…”