Sodium deoxycholate-based protocol (SDC)

The SDC protocol was first described by Wakabayashi et al.¹¹. The deparaffinized tissues were incubated with 80 µL buffer containing 2% SDC and 1 mM EDTA in 50 mM Tris in a thermoshaker for 2 h at 95 °C, treated with a Bioruptor Pico sonicator (20 cycles on high, 30 s on/off), and centrifuged for 10 min at 127,000 × g. An aliquot of the supernatant was used for protein level BCA. Samples were reduced and alkylated with 12.5 mM DTT and 55 mM IAA and then diluted 1:5 with Tris and then pre-digested with LysC at 25 °C and digested overnight with trypsin at 37 °C (enzyme:substrate ratio 1:50). After digestion, ethyl acetate was used to remove the remaining paraffin, and the samples were acidified with 100% formic acid. Insoluble particles were spun down and the peptides were desalted on C18 material. An aliquot of the desalted peptides was used for a peptide level BCA.