Lysate preparation, Click chemistry, Trypsin digestion, Peptide enrichment, and DTT/IAA elution

For cell surface lysine reactivity detection, the light cells were labeled by 50 µM OPA-S-S-alkyne and the heavy cells were labeled by 500 µM OPA-S-S-alkyne in the forward SILAC experiments. In the reverse SILAC experiments, the heavy cells were labeled by 50 µM OPA-S-S-alkyne and the light cells were labeled by 500 µM OPA-S-S-alkyne. After cell lysis and protein concentration determination, the light and heavy lysates (500 µg each) were combined and incubated in 500 µL DPBS buffer containing 100 μM biotin-PEG₄-azide, 2.5 mM freshly prepared sodium ascorbate, 500 µM BTTP, 250 µM CuSO₄ for 2 h at room temperature with rotation.

The click-labeled lysates were precipitated by 2 mL cold acetone at −20 °C overnight. The precipitated proteins were centrifuged at 15,000 × g for 30 min at 4 °C and washed twice with 1 mL cold methanol. Protein pellets were resuspended in 1 mL 50 mM ammonium bicarbonate and trypsin (enzyme: substrate ratio was 1:50) was then added. Trypsin digestion was performed at 37 °C with rotation overnight. Then 50 µL neutravidin beads (prewashed three times with PBS) were added to the solution, and the resulting mixture was incubated for 3 h at room temperature with rotation. The beads were pelleted by centrifugation and washed with 0.1% SDS in PBS three times, 8 M urea in PBS three times, 10 × PBS three times and distilled water three times to remove nonspecifically bound peptides. The modified peptides were released from the beads by two treatments of 100 µL 50 m M DTT at 37 °C for 30 min with rotation to cleave the disulfide bridge in the labeling reagents. The beads were then washed with 50% (v/v) ACN/H₂O containing 0.1% TFA, and the washes were combined with the eluent to form the cleavage fraction. The labeled peptides were desalted with C18 Zip-Tips (Merck) and then incubated with 100 mM iodoacetamide for 45 min at room temperature in the dark. The resulting peptides were desalted with C18 Zip-Tips and dried in a vacuum centrifuge.