MALDI2-MSI (Michellod et al. 2023)

<em>Olavius algarvensis</em> specimens were prepared following Kadesch et al. (2019) with a few modifications. Briefly, 20 µL 6.7% glutaraldehyde solution in marine phosphate buffered saline were deposited on a glass slide. Gutless annelids were transferred to the fixative using bent acupuncture needles. A coverslip was applied, and samples were frozen in liquid nitrogen and stored at −80°C until further processing. A single worm was transferred using featherweight forceps onto a sodium carboxymethyl cellulose (CMC; Sigma-Aldrich) stamp which was glued onto a cryostat specimen disc (Leica). Gelatin solution (8 % (β = 80 g L-1), 10-20 µL) was used to coat the worm. The specimen disc with the sample was transferred to the cryostat (Leica CM3050 S, Leica Biosystems) and kept for 30 min at -22°C before sectioning into sections of 12 µm thickness (chamber temperature -22°C, object temperature -22°C). Sections were thaw-mounted on IntelliSlides (Bruker Daltonics), and their quality determined microscopically. Sections of sufficient quality were stored in a desiccator at room temperature (RT) until further analysis. 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) (<em>4</em>) as described in (<em>5</em>). The mass resolving power of this hybrid QTOF-type instrument is about 40,000 (fwhm) in the investigated <em>m/z</em> range of 300–1500. For material ejection, a scan range of 1 μm of the laser spot on the target was used, resulting in an ablated area of 5 μm in diameter. The step size of the stage during the MSI run was set to 5 μm. The laser power was set to 40%, with 50 laser shots/pixel.