Proteasomal Degradation of S100-β Protein

Purified human S100-β (23 (link)) was incubated with purified 20S proteasome (Enzo Life Sciences, Farmingdale, NY, USA) (molar ratio 250:1) in digestion buffer [30 mM Tris-HCl (pH 8.0), 10 mM NaCl, 2 mM MgCl₂, 1 mM DTT, 0.01% sodium dodecyl sulfate) for 16 h at 37°C. As a control, the same digestion was set up with either no proteasome or with acetic acid (1%)–inactivated proteasome. Generated peptides were purified and concentrated using a cationic resin (ZipTip with strong cation exchange; MilliporeSigma) following the manufacturer’s instructions. Retained peptides were eluted from the resin and analyzed by MS.
The S100-β human protein sequence was analyzed in silico for potential proteasome and immunoproteasome cleavage sites using several published algorithms (26 (link), 27 (link)).