Ito glass slide

Fresh frozen mouse brains were cut at a thickness of 12 μm using a cryostat microtome (Leica CM, Leica Microsystems). Tissue sections were thaw-mounted onto conductive indium tin oxide (ITO) glass slides (Bruker Daltonics) and stored at −80 °C. Sections were desiccated at room temperature for 15 min before spray coating of norharmane matrix solution. Prior to matrix coating, the slide was scanned on a flatbed scanner (Epson perfection V500). The matrix solutions were prepared by dissolving the norharmane matrix powder in 80% MeOH (7.5 mg/ml) solution in a glass vial and sonicated briefly. An automated pneumatic sprayer (HTX-Technologies LLC, Chapel Hill, NC, USA) was used, which was combined with a pump (AKTA FPLC P-905 pump, Cytiva, Uppsala, Sweden) to spray heated matrix solution over the tissue sections. The pump was kept running at 100 μL/min using a 50% acetonitrile pushing solvent before the experiments to ensure a stable flow of the solvent with isocratic pressure. The matrix solution was sprayed using instrumental parameters of a solvent flow rate of 70 μL/min at isocratic pressure, a nitrogen flow of 6 psi, spray temperature of 60 °C, 15 passes with offsets and rotations, a nozzle head velocity of 1200 mm/min, and track spacing of 2.0 mm.

E. fetida sections were mounted onto ITO glass slides (Bruker Daltonik, GmbH), and the slides were scanned using an Epson Perfection V500 scanner (Epson Europe, Amsterdam, Netherland) with a resolution of 2400 DPI. Subsequently, HCCA was prepared (7 mg/mL in 50% acetonitrile and 0.2% trifluoroacetic acid) and sprayed using ImagePrep (Bruker Daltonik, GmbH).
The mass spectrometry experiments were performed on a MALDI-TOF/TOF Bruker ultrafleXtreme mass spectrometer (Bruker Daltonik, GmbH). The scanning raster was set to 50 μm, and prior to each measurement, the mass spectrometer was calibrated using a standard calibration mixture of proteins and peptides (Bruker). For low-mass molecules, imaging mass spectrometry (IMS) was performed in reflectron positive mode, and for metallothioneins the IMS was performed in linear positive mode; both analyses were performed at 45% laser power. The MS spectra were typically acquired after averaging 500 subspectra from a total of 500 laser shots per raster spot. After analyses, the mass spectra were automatically loaded into flexAnalysis and subsequently processed (baseline subtraction).
Moreover, IMS figures were prepared after selecting the peaks of interest—PC₂/PC₃, (E₂)-3,4-Q, GSSG and MTs. The molecular weights were selected according to the UniProt database (www.uniprot.org) and processed in a molecular weight + hydrogen ± 0.05% format.

The following solvents were used for processing of tissue sections on ITO glass slides: xylene (mixture of isomers) pure per analysis (POCh, Gliwice, Poland), ethanol HPLC grade (POCh, Gliwice, Poland), demineralized water from a Simplicity UV system equipped with an LC-Pak Polisher filter (both from Millipore SAS, Molsheim, France). Sequencing Grade Modified Trypsin from Promega (Madison, WI, USA) was used as a proteolytic enzyme. Ammonium bicarbonate BioUltra was purchased from Sigma Aldrich (St. Louis, MO, USA), acetonitrile (ACN), methanol (MeOH) and trifluoroacetic acid (TFA)—all HiPerSolv CHROMANORM for LC-MS grade—were obtained from VWR Chemicals (Radnor, PA, USA). ITO glass slides, alpha-cyano-4-hydroxycinnamic (HCCA) and Peptide Calibration Standard II were purchased from Bruker Daltonik (Bremen, Germany). Poly-l-lysine solution (0.1% w/v in H₂O) and IGEPAL^® CA-630 were both obtained from Sigma-Aldrich. ITO glass slides were coated with poly-l-lysine according to a procedure given in [20 (link)]. RapiGest SF Surfactant and TopTips (10–200 µL) C18 were purchased from Waters (Milford, MA, USA) and Glygen Corp. (Columbia, MD, USA), respectively.