HEK 293 cells stably expressing cat caspase-12 with a Strep-tag or empty control were lysed in Buffer W (Strep-tag protein purification buffer, IBA) supplemented with 0.1% Triton X-100 and 5 mM MgCl2. As Buffer W contains 1 mM EDTA, MgCl2 was added to block EDTA except for the sample 4 “With EDTA Bait”. The cell lysates (from 10 × 100 mm dishes) were incubated with or without Pam3CSK4 (10 μg) at 37°C for 30 min in the presence or absence of 5 mM ATP. Cat caspase-12 was enriched by affinity chromatography using Strep-tactin resin columns, and eluted proteins from the columns were subjected to LC-MS/MS analysis. After acetone precipitation, the proteins were dissolved in 6 M Urea and 50 mM TEAB (triethylammonium bicarbonate) pH8.5. Proteins (900 ng) were adjusted to a final volume of 10 μL, reduced with 5 mM TCEP for 30 min at 37°C in the dark, alkylated with 24 mM iodoacetamide for 30 min at room temperature in the dark. Alkylated proteins were digested with trypsin (Thermo Fisher Scientific) at a 1:10 enzyme/protein ratio for 16 h at 37°C. Peptides were desalted with Stage tip #84850 (Thermo Pierce, Tokyo, Japan) and eluted with 70% ACN. Then, eluted peptides were dried by vacuum centrifuge and dissolved with 5% ACN containing 0.1% trifluoroacetic acid. The trypsin-digested peptides were analyzed by Orbitrap QE plus (Thermo Fisher Scientific) with nano-liquid chromatography (EASY-nLC 1200; Thermo Fisher Scientific). The purified peptides were loaded and separated on the column (25 cm x 75 μm ID, 1.6 mm C18; Ionoptics) with a linear acetonitrile gradient (0-40%) in 0.1% formic acid at a flow rate of 300 nL min-1. The peptide ions were detected by Orbitrap QE plus MS; Thermo Fisher Scientific) in the data-dependent acquisition mode with the installed Xcalibur software (Thermo Fisher Scientific). Full-scan mass spectra were acquired in the MS over 375-1,500 m/z with resolution of 70,000.