Mass spectrometry analysis

After Click reaction, proteins were precipitated using methanol. The washed protein pellets were dissolved in resuspension buffer containing 4% (w/v) SDS, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl and 10 mM EDTA. Proteins were diluted with 1 volume of Brij97 buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM EDTA, 1% Brij97). Prewashed streptavidin-agrose beads were added to each sample. The protein and beads were incubated for 1.5 hours at room temperature on a rotator. The beads then were washed three times with 0.2 SDS in PBS and once with 250 mM ammonium bicarbonate. The captured proteins were incubated for 40 min in dark with 500 μL 8M urea, 50 μL 500 mM TCEP and 50 μL of 400 mM iodoacetamide and then washed twice with 250 mM ammonium bicarbonate. The samples were directly digested on beads and supernatants were collected for LC-MS/MS analysis as described previously¹⁹.