Sequencing grade modified

Protein in-gel trypsin was digested manually (sequencing grade modified, Promega). Selected spots with differential expression were manually cut out from reference gels and were washed sequentially with 25 mM ammonium bicarbonate (NH₄HCO₃) and acetonitrile (ACN). The proteins were reduced with 20 mM DTT solution for 60 min at 60°C and alkylated with a 50 mM solution of iodine acetamide for 30 min at room temperature. After sequential washings with buffer and acetronitrile, the proteins were digested overnight at 37°C with 80 ng of trypsin. Peptides were extracted from the gel matrix with 10% formic acid (FA) and can, pooled and dried in a vacuum centrifuge. The trypsin-digested peptide samples were analyzed by LC-MS/MS.

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.

Histone extracts were prepared according to published protocol [32 (link)]. Protein concentration was measured with Bradford Assay (Bio-Rad, Herkules, CA, USA). For chemical derivatization, aliquots of 12 μg of histone extract were used. Microwave-assisted histone derivatization using trimethylacetic anhydride and trypsin digestion (Sequencing grade modified, Promega Corporation, Madison, WI, USA) were performed according to a previously published procedure [29 (link)]. The detailed protocol for histone preparation prior to LC-MS/MS is enclosed in the Supplementary Methods.