Human lung fibrosis samples. Mouse models of lung fibrosis in WT and Epm2a, and Gaa knockout strains were analyzed using MALDI-MSI. Analytes analyzed were N-linked glycans and glycogen.

Tissues were sectioned at 4 μm and mounted on positively charged glass slides for MALDI imaging as previously described. Slides were heated at 60°C for 1 hr. After cooling, tissue sections were deparaffinized by washing twice in xylene (3 min each). Tissue sections were then rehydrated by washing slides twice in 100% ethanol (1 min each), once in 95% ethanol (1 min), once in 70% ethanol (1 min), and twice in water (3 min each). Following washes, slides were transferred to a coplin jar containing citraconic anhydride buffer for antigen retrieval and the jar was placed in a vegetable steamer for 25 min. Citraconic anhydride buffer was prepared by adding 25 µL citraconic anhydride in 50 mL water and adjusted to pH 3.0 with HCl. After antigen retrieval, slides were dried in a vacuum desiccator prior to enzymatic digestion. An HTX spray station (HTX) was used to coat the slide with a 0.2 ml aqueous solution of isoamylase (3 units/slide) and PNGase F (20 mg total/ slide). The spray nozzle was heated to 45˚C with a spray velocity of 900 m/min. Following enzyme application, slides were incubated at 37˚C for 2 hr in a humidified chamber, and dried in a vacuum desiccator prior to matrix application [a-cyano-4-hydroxycinnamic acid matrix (0.021 g CHCA in 3 ml 50% acetonitrile/50% water and 12 µL 25%TFA) applied with HTX sprayer]. For detection and separation of glycogen and N-glycans, a Waters SynaptG2-Si high-definition mass spectrometer equipped with traveling wave ion mobility was used. The laser was operating at 1000 Hz with an energy of 200 AU and spot size of 75 µm, mass range is set at 500–3000m/z. Ion mobility settings were done according to previously established parameters with a trap entrance energy of 2V, trap bias of 85V, and DC/exist of 0V. Wave velocity settings were set to: trap 9.6 m/s, IMS 4.6m/s, transfer 17.4 m/s. Wave height settings were set to: trap 4V, IMS,42.7, transfer 4V, and additional settings are variable wave ramp down of 1400 m/s. Data was acquired with Masslynx v4.2. MALDI images were produced using the HDI software v1.5 (Waters Corp) following built-in peak integration function to account for mass drift over the MALDI run. All MALDI images were normalized to total ion current (TIC) within each pixel.