Caper Affinity Protein Purification

Caper affinity purifications were denatured, reduced and alkylated using 5% (w/v) sodium dodecyl sulfate (SDS), 10 mM tris(2-carboxyethylphosphine) (TCEP), 40 mM 2-chloroacetamide, 50 mM Tris–HCl, pH 8.5 with boiling 10 min, then incubated shaking at 1000 rpm at 37°C for 30 min. Proteins were digested using the SP3 method (Hughes et al., 2014 (link)). Briefly, 200 μg carboxylate-functionalized speedbeads (Cytiva Life Sciences) were added followed by the addition of acetonitrile to 80% (v/v) inducing binding to the beads. The beads were washed twice with 80% (v/v) ethanol and twice with 100% acetonitrile. Proteins were digested in 50 mM Tris–HCl, pH 8.5, with 0.5 μg Lys-C/Trypsin (Promega) and incubated at 37°C overnight. Tryptic peptides were desalted with the addition of 95% (v/v) acetonitrile binding the peptides back to the beads and washed once with 100% acetonitrile. Peptides were collected from the beads with two elutions of 1% (v/v) trifluoroacetic acid, 3% (v/v) acetonitrile. Cleaned-up peptide were then dried in a speedvac vacuum centrifuge and stored at −20°C until analysis.