Affinity Purification of Sulfo-NHS-SS-Biotin Labeled Peptides

Sulfo-NHS-SS-biotin labelled peptides were immobilized on high capacity neutravidin agarose (Thermo Scientific). The binding efficiencies of the column were examined by dot-blot experiments (Supplementary Methods), in order to determine the optimal volume of neutravidin agarose (100 µl/sample). Labelled mixtures were incubated on affinity columns for 1 hour at room temperature. Columns were washed extensively to reduce the number of non-specific peptides or contaminants as described by Lango et al.^{43 (link)}. The covalently modified peptides were eluted from the column with 50 mM DTT in 50 mM NH₄HCO₃ (pH = 8) for 1 hour at 37 °C, which were followed by an alkylation step with 110 mM iodoacetamide in dark at room temperature.