Fleximaging 2

The molecular characterization was performed with MS/MS and a MALDI linear quadrupole ion-trap mass-spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The MS/MS analysis was performed directly on the brain sections. The acquisition was in the mid-mass-range mode (m/z 100–1,000), which is the positive-ion detection mode, with an ionization voltage of 30 V and a collision voltage of 35 V. The PC molecular species were identified from the neutral loss compositions, which were determined from the deltas of the precursor and product ions in the MS/MS spectra.
The IMS of the identified PC species was performed with a MALDI time-of-flight (TOF)/TOF-type instrument (Ultraflex II TOF/TOF; Bruker Daltonics GmbH) that was equipped with a 355-nm Nd:YAG laser. The data were acquired in the positive-ion reflector mode under an accelerating potential of 20 kV. Calibration was performed with an external calibration method. The signals of m/z = 700–1,000 were measured. Raster scans on the tissue surfaces were performed automatically with FlexControl and FlexImaging 2.0 software (Bruker Daltonics GmbH). The number of laser irradiations was 200 shots per spot. Image reconstruction was performed with FlexImaging 2.0 software.

The tissue sections were analyzed using a matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) type instrument, Ultraflex II TOF/TOF (Bruker Daltonics), which was equipped with a 355 nm smartbeam laser at 200 Hz repetition. Data were acquired in the positive-ion mode using an external calibration method with ions from DHB, angiotensin II, and bradykinin, which covered a mass-to-charge ratio (m/z) range of 100 to 1200. Calibration proteins were deposited on the surfaces of sample sections. Each raster scan was automatically performed in the regions of cancer and normal tissue. The interval between data points was 50 μm, and 100 laser beam shots in a random walk mode for each data point were irradiated. The mass spectrometry parameters were manually optimized to obtain the highest sensitivity, with m/z values in the range of 500–900. All mass spectra were acquired automatically using FlexImaging 2.1 software (Bruker Daltonics).

A part of each frozen testis (used for histology) was sectioned at 10 μm of thickness with a cryostat (CM 1950, Leica Microsystems). The sections were thaw-mounted onto indium tin oxide (ITO)-coated slides (Bruker Deltonics, Bremen, Germany), dried and then kept at -30°C until IMS analysis. Before IMS analyses, the sections were dried at room temperature and then sprayed with matrix solution using a 0.2-mm nozzle caliber airbrush (Procon Boy FWA Platinum, Tokyo, Japan). The matrix used was 2,5-dihydroxybenzoic acid (DHB) (Bruker Daltonics), and it was firstly dissolved to reach a concentration of 50 mg/ml in 70% methanol and 0.1% trifluoroacetic acid (TFA). A calibration process was performed using 10 pmol/μl bradykinin and 10 pmol/μl human angiotensin-II as standard peptides by applying on to the sprayed area out of the tissue sections. The sprayed sections were then analysed in a positive ion mode using an ultraflex II MALDI TOF/TOF mas nics). The mass spectra were obtained in the mass ranges between m/z 500–1000. The settings of laser s spectrometer (Bruker Delto irradiation were 200 Hz frequency and a raster width at 20 μm. After IMS analyses, ion images were obtained using flexImaging 2.1 software (Bruker Daltonics). Finally, the analysed sections were stained with H&E to confirm the histology of the area of interest.

The MALDI matrix was applied using an ImagePrep device as described above. Protein imaging was then performed on an Ultraflex II MALDI-TOF/TOF instrument (Bruker Daltonics, Bremen, Germany) equipped with a smartbeam laser (Nd:YAG, 355 nm). Protein mass spectra were acquired in linear positive ion mode within a mass range of m/z 3,000–30,000. The distance between raster points was set to 80 μm with a total of 500 laser shots accumulated at a 200-Hz repetition rate for each pixel. Spectra were processed by baseline correction and smoothed using FlexAnalysis 3.2 software (Bruker Daltonics, Bremen, Germany). Image analysis and data visualization were performed with FlexImaging 2.1 software (Bruker Daltonics, Bremen, Germany). For statistical analysis, datasets obtained from FlexImaging were loaded into ClinProTools 2.2 software (Bruker Daltonics, Bremen, Germany) to conduct hierarchical clustering. Unsupervised clustering was selected with Euclidean as the distance method and Ward as the linkage method.

We used a MALDI-TOF-TOF-type instrument (ultraflex II TOF/TOF, Bruker Daltonics) to analyze the tissue sections. An Nd:YAG laser (355-nm wavelength) was used for the desorption and ionization of PLs with a 200-Hz repetition rate. The setting parameters for the laser energy, detector gain, and random walk function were optimized manually for each measurement to obtain the highest sensitivity for the m/z range of 460–1,000. The calibration of m/z values detected in each IMS measurement was performed by using Mono DHB ([M + H]⁺, m/z 155.03), bradykinin ([M + H]⁺, m/z 757.40), and angiotensin II ([M + H]⁺, m/z 1046.54) in the positive ion mode. The measurement regions in the War-T and Non-T tissues were automatically scanned by the laser using flexControl (Bruker Daltonics). The number of laser shots at each measurement spot was 200, and the intervals between each measurement point were 200 μm. All data were acquired and visualized by flexImaging 2.1 software (Bruker Daltonics).

All MSI measurements were performed in an Autoflex III MALDI-TOF/TOF spectrometer (Bruker Daltonik) equipped with a Smartbeam laser (355nm, 200 Hz) and controlled using the Flex Control 3.0 software package. The mass spectrometer was operated with positive polarity in reflectron mode, and spectra were acquired in the range of 500–2000 m/z.
The laser raster size was set at 100 μm, which is approximately equal to the laser spot diameter. At this resolution, it takes approximately 30min to complete an image of one wheat section. The signal was initially optimized by manually adjusting the laser power and the number of laser shots fired. According to this procedure, full-scan MS experiments were run with 200 laser shots per step and using the laser power that generated the best signal-to-noise ratio. Image acquisition at tissue surfaces was performed using Flex Imaging 2.1 software (Bruker Daltonik). Relative comparisons of the released oligosaccharides in the different tissue sections and of the ratios of different ions in the same tissue section were performed through labelled normalization using MALDI Tools 1.1 software (Källback et al., 2012 (link)) compatible with Flex Imaging 2.1.