Formalin-fixed paraffin embedded (FFPE) lungs were processed as previously described.15 (link),16 (link) In brief, FFPE lungs were sectioned at 5 µm on glass slides. Tissues were dewaxed and rehydrated followed by an antigen retrieval process in citraconic anhydride buffer. Recombinant PNGase F (0.1 µg/mL) was applied by a M5 TM robotic Sprayer (HTX Technologies LLC, Chapel Hill, NC). Following incubation in a humidity chamber for 2 hours, slides were desiccated for 24 hours. The following day, 7 mg/mL of alpha-Cyano-4-hydroxycinnamic acid in 50% acetonitrile with 0.1% TFA were applied to each slide by the robotic sprayer. Slides were subsequently stored in a desiccator prior to MALDI-MSI analysis.
A Waters Synapt G2Si mass spectrometer (Waters Corporation, Millford, MA) equipped with a Nd:YAG UV laser with a spot size of 100 µm was used to detect N-glycans at X and Y coordinates of 100 µm. Following data acquisition, mass spectra were analyzed by High Definition Imaging Software (Waters Corporation) for mass range 500–3500 m/z. Three regions of interest were determined for each lung; pixel intensities were averaged and normalized by total ion current. Representative N-glycans were generated by Glycworkbench.
A Waters Synapt G2Si mass spectrometer (Waters Corporation, Millford, MA) equipped with a Nd:YAG UV laser with a spot size of 100 µm was used to detect N-glycans at X and Y coordinates of 100 µm. Following data acquisition, mass spectra were analyzed by High Definition Imaging Software (Waters Corporation) for mass range 500–3500 m/z. Three regions of interest were determined for each lung; pixel intensities were averaged and normalized by total ion current. Representative N-glycans were generated by Glycworkbench.
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