Sodium dodecyl sulfate-based protocol (SDS-SP3)

The protocol was first described by Hughes et al.¹⁰. Here, the deparaffinized tissues were incubated with 40 µL buffer containing 50 mM Tris, 1% SDS, and 2 µL benzonase for 30 min at 37 °C. Afterwards, 40 µL buffer containing 2% SDS and 5 mM DTT were added and the samples were incubated 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. After alkylation with 50 mM IAA, the remaining IAA was blocked with 113 mM DTT. 10 µL mix of paramagnetic beads containing 1:1 hydrophilic and hydrophobic beads were added for a concentration of 1 µg protein:10 µg beads. The concentration of organic solvent was increased to >70% by the addition of 800 µL acetonitrile and the proteins were incubated with the beads first on the bench and then in the magnetic rack. After the beads had settled, the supernatant was removed, and the beads were washed three times with 70% ethanol. Proteins were eluted from the beads with 100 µL 50 mM HEPES, pH 8. Digestion was performed overnight with LysC and trypsin (enzyme:substrate ratio 1:50). The next morning, samples were again incubated on the magnetic rack and the bead-free supernatant was transferred to fresh tubes and the beads were washed again with 100 µL HEPES. Samples were acidified with 100% formic acid, and desalted on C18 material. An aliquot of the desalted peptides was used for a peptide level BCA.