Smartbeam 2 nd yag 355 nm laser

Post enzyme releasing steps, α-Cyano-4-hydroxycinnamic acid, CHCA (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 7 mg/ml (50% ACN with 0.1% TFA) was sprayed over the tissue sections using the TM-Sprayer with the following settings: 100 μl/min, 10 passes, crisscross pattern, a velocity of 1,300 mm/min, 3 mm track spacing. MSI experiments were performed using a 15 Tesla SolariX Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a dual ESI/MALDI ion source and a Smart-beam II Nd:YAG (355 nm) laser. The instrument was operated in positive ion mode over an m/z range of 1,000–5,000 with an estimated resolving power of 330,000 at m/z 400. The target plate stepping distance was set to 50 μm, which defined the spatial resolution for these measurements (i.e., the laser spot size was smaller than the stage stepping distance). Higher spatial resolution imaging experiments (at 15 μm spatial resolution) were performed using a MALDI source (Spectroglyph, LLC, Kennewick, WA, USA) with a 1 kHz Explorer One Nd:YAG (349 nm) laser (Spectra-Physics, Santa Clara, CA, USA) coupled to a custom ThermoFisher QE-Orbitrap MS (Bremen, Germany) (Zemaitis et al., 2022 (link)) in positive ion mode in an m/z range of 600–6,000 with a mass resolution of ~200,000 at m/z 400.

Tissue imaging via laser desorption/ionization–mass spectrometry (LDI–MS) imaging was performed as previously described [21 (link),68 (link),69 (link)] in 10 µm heart sections covered by gold monolayers. LDI–MS tissue images were acquired using a MALDI-TOF UltrafleXtreme instrument with SmartBeam II Nd:YAG/355 nm laser (Bruker Daltonics, Billerica, MA, USA) and processed by using open-source software rMSI (version 0.3.1) [70 (link)] and rMSIproc (https://github.com/prafols/rMSIproc (version 0.9.1) (accessed on 2 July 2022)) [69 (link)]. The metabolites’ tentative identification was based on the exact mass of their sodium adduct according to the Human Metabolome Database, filtering metabolites with mass error Δ < 50 ppm, and the results obtained from the LC–MS/MS experiments.

One image of a sagittal brain tissue section and six liver tissue sections (three slices from a control animal and three sections from a THS-exposed animal) were acquired using a MALDI TOF/TOF UltrafleXtreme instrument with SmartBeam II Nd:YAG/355 nm laser from Bruker Daltonics, also at the COS facilities. Raster sizes of 80 and 20 µm were used for the brain and liver tissue sections, respectively. The TOF spectrometer operated in reflectron positive mode with the digitizer set at a sample rate of 1.25 GHz in a mass range between 70 and 1200 Da. The spectrometer was calibrated prior to tissue image acquisitions using [Au]⁺ cluster MS peaks as internal mass references [27 (link)].

A MALDI TOF/TOF ultrafleXtreme instrument with SmartBeam II Nd:YAG/355 nm laser from Bruker Daltonics available at COS was used for MSI acquisition. Acquisitions were carried out by operating the laser at 2 kHz and collecting a total of 500 shots per pixel.
The TOF spectrometer was operated in positive ion, reflectron mode, in m/z ranges according to Table 1. The spectrometer was calibrated prior to MSI data acquisition using ${\left[\mathit{Ag}\right]}_n^{+}$ cluster peaks as internal reference masses.