Fleximaging 4

An ImagePrep spray station (Bruker Daltonics, Billerica, MA, USA) was used to coat the slide with a 0.2 mL aqueous solution of PNGaseF (20 µg total/slide) as previously described [21 (link)]. As negative control, adjacent control tissue slices were shielded from PNGaseF application by covering the tissue section with a glass slide. Digestion occurred in a humidified chamber at 37 °C for 2 h. Slides were desiccated prior to α-cyano-4-hydroxycinnamic acid matrix application (0.021 g CHCA in 3 mL 50% acetonitrile/50% water and 12 µL 25% TFA) using the ImagePrep sprayer. Released glycan ions were detected using a Solarix dual source 7T FTICR mass spectrometer (Bruker Daltonics) (m/z 690–5000) with a SmartBeam II laser operating at 1000 Hz, a laser spot size of 25 μm. Following MS analysis, data was loaded into FlexImaging Software focusing on the range m/z = 1000–4000 and reduced to 0.95 ICR Reduction Noise Threshold. FlexImaging 4.0 (Bruker Daltonics) was used to generate images of differentially expressed glycans. Observed glycans were searched against the glycan database generated using GlycoWorkbench [25 (link)]. Presented glycan structures were generated in GlycoWorkbench and represent putative structures determined by combinations of accurate m/z and off-slide derivatization experiments. CASI/CID was done as previously described [20 (link),21 (link)].

Mass spectra were acquired using a 9.4T SolariX Fourier transform mass spectrometer (Bruker Daltonics, Billerica, MA). Images were acquired with a pixel step size for the surface raster set to 150 µm in FlexImaging 4.0 (Bruker Daltonics, Billerica, MA). Using serially sectioned tissue samples one image was acquired in positive ion mode and a second image acquired in negative ion mode. Laser power was set to 20% for positive mode and 15% for negative mode. Acquisition range was set to m/z 100–3000, 10 positions were sampled (25 shots/position at a laser frequency of 1000 Hz) within a given pixel for a total of 250 laser shots per pixel.

Fourty microns-thick transverse frozen sections were cut using a cryostat (Leica, Milton Keynes, UK) and fixed on a carbon-conductive adhesive tape which was in turn fixed on an indium tin oxide (ITO) slide (Bruker Daltonics, Bremen, Germany, cat no 237001).
All MSI measurements were performed using an Autoflex-Speed MALDI-TOF/TOF spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam laser (355 nm, 1000 Hz) and controlled using the Flex Control 3.4 software package. The mass spectrometer was operated with a negative polarity in the reflectron mode. Spectra were acquired in the mass range of m/z 100–600 for all (x, y) coordinates corresponding to the imaged tissue.
The laser raster size was set at 50 microns. The signal was initially optimized by manually adjusting the laser power and the number of laser shots fired. Accordingly, full-scan MS experiments were run by accumulating 400 satisfactory laser shots per raster position, and using the laser power leading to the best signal-to-noise ratio. Image acquisition was performed using the Flex Imaging 4.0 (Bruker Daltonics) software package. The correlation of the target plate with the optical image was performed from three distinct teaching points following the procedure of the Flex Imaging software (Bruker Daltonics).

Samples preparation and data acquisition were performed under the same conditions as previously described by Gadea et al. [21 (link)]. Briefly, Pseudocyphelaria crocata samples were hand-cut using a razor blade to afford slices approximately 100 μm thick. Lichen slices were fixed on a carbon-conductive adhesive tape that was, itself, fixed on an indium tin oxide (ITO) slide (Bruker Daltonics, Bremen, Germany, cat. no. 237001). All MSI measurements were performed using an Autoflex-Speed MALDI-TOF/TOF spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam laser (355 nm, 1000 Hz) and controlled using the Flex Control 3.4 software package. The mass spectrometer was used in the reflectron mode with a negative polarity. Spectra were acquired in the mass range of m/z 100 to 1000 for all (x, y) coordinates corresponding to the imaged tissue. The laser raster size was set at microns. The signal was initially optimized by manually adjusting the laser power and the number of laser shots fired. Accordingly, full-scan MS experiments were run by accumulating 400 laser shots per raster position and by using the laser power leading to the best signal-to-noise ratio. Image acquisition was performed using the Flex Imaging 4.0 (Bruker Daltonics) software package.