Pierce streptavidin beaded agarose resin

Labelled proteome samples were diluted to 0.2% SDS with PBS. The resulting samples were then added to 100 µl of Pierce streptavidin beaded agarose resin (Thermo Scientific) and incubated overnight at 4 °C followed by a further 2 h at room temperature. Protein-bound beads were washed with 1× 0.2% SDS in PBS, 3× PBS and 3× H₂O before resuspending in 6 M urea in PBS + 10 mM DTT and incubating at 65 °C for 15 min. Reduced samples were then alkylated by adding IAA to a final concentration of 20 mM and incubating for 30 min at 37 °C with rotation. Samples were diluted three-fold with PBS and centrifuged (1,400g, 2 min) to pellet the beads. The beads were resuspended in a mixture of 200 µl of 2 M urea in PBS, 1 mM CaCl₂ and 2 µg trypsin (Promega) and incubated overnight at 37 °C. The beads were separated from the digest by centrifugation and washed three times with PBS and three times with H₂O. Azo-labelled peptides were then cleaved by adding 50 mM sodium hydrosulfite (Na₂S₂O₄) and rotating at room temperature for 1 h. Eluted peptides were then collected from the supernatant, and Na₂S₂O₄ cleavage was repeated twice more to fractionate the sample. Between each cleavage, the beads were washed with 2× H₂O and combined with the previous elution. Formic acid was added to the sample to 20% (v/v) concentration before storing at −20 °C until MS analysis.