Chip 1000

On localized pre-neoplastic lesions, tryptic digestion was performed using a Chemical Inkjet Printer (CHIP-1000, Shimadzu, Kyoto, Japan). The region was carefully selected to ensure that the analysis was restricted to the epithelial cells marked by IHC, thus reducing the potential for contamination from other cell types. The trypsin solution (Sequencing grade modified (Promega), 40 µg/mL, 50 mM NH4HCO3 buffer) was deposited on a region defined to 600 × 600 µm² during 2 h. During this time, the trypsin was changed every half-hour. With 350 cycles and 450 pL per spot, a total of 6.3 µg of trypsin was deposited. To stop digestion, 0.1% TFA was spotted during 25 cycles.

A chemical inkjet chip printer (Shimadzu CHIP-1000) was used for printing enzymes to tissue surfaces (the workflow is shown in supplemental Fig. S3). This device is capable of applying minimal 500 pL solutions to tissue surfaces. The printer scans an image of the TMA slide, which enables the user to designate the cores to be analyzed. Next, 1 μl enzyme solution, sufficient to cover the entire core surface, was applied to each designated core using a user-defined printing pattern. To ensure exhaustive digestion, the enzyme printing process was repeated four times. The time interval for the repeated enzyme printing was 2 h. After the fourth enzyme printing, the TMA slides were incubated in a humidified digestion chamber at 37 °C, overnight. The detailed on-slide digestion protocol could be obtained from our previous publications (27 (link), 28 (link)). Briefly, a mixture of 0.5 mU/μl heparin lyases I, II, and III was applied first, followed by overnight incubation. Second, a 1 mU/μl chondroitinase ABC enzyme solution was applied to the same spot. Finally, 100 ng/μl trypsin solution was applied to the spots.

Four sample slides were placed in a slot on a MALDI target plate and attached with conductive tape. The prepared matrix solution was 2,5-dihydroxybenzoic acid (50 mg/mL) in 50% methanol and 0.05% trifluoroacetic acid. The matrix solution was added to the sections using a CHIP-1000 chemical inkjet printer (Shimadzu, Kyoto, Japan) with a droplet size of 5-nL by micro-spotting in 25 cycles of 200 pL per spot at a spatial distance of 250 μm. After spotting, the target plate was dried in a desiccator at 20 °C.