Azide-Alkyne Click Chemistry Protocol

Cells were lysed by heating in 1% SDS in PBS at 90 °C for 10 min and lysates were cleared by centrifugation. Protein concentrations were determined with the BCA protein quantitation kit (Thermo Scientific), and paired ‘light’ and ‘heavy’ cultures were mixed at equal quantities of total protein. Azide-alkyne click chemistry was performed as described in Hong et al. with a 0.1 mM alkyne-DADPS tag and allowed to proceed for 4 hr at room temperature (Fig. S1e).³⁴ The DADPS tag was synthesized as described previously.^{35 (link)} Proteins were concentrated by acetone precipitation and solubilized in 2% SDS in PBS. Solutions were diluted to 0.15% SDS in PBS, and tagged proteins were captured by incubating with Streptavidin UltraLink Resin (Thermo Scientific) for 30 min at room temperature. Resin was washed with 35 column volumes of 1% SDS in PBS and 10 column volumes of 0.1% SDS in ddH₂O. The DADPS tag was cleaved by incubating the resin in 5% formic acid in 0.1% SDS in ddH₂O for 1 hr. Columns were washed with 5 column volumes of 0.1% SDS in H₂O, during which proteins remained bound, and proteins were subsequently eluted in 15 column volumes of 1% SDS in PBS. Protein enrichment was confirmed by SDS-PAGE, and eluted proteins were concentrated on 3kDa MWCO spin filters (Amicon).