Prox-SILAC labeling in cells

For SILAC medium preparation, L-arginine and L-lysine were replaced by heavy-labeled L-arginine-¹³C₆-¹⁵N₄ and heavy-labeled L-lysine-¹³C₆-¹⁵N₂. Each L-arginine-¹³C₆-¹⁵N₄ introduces +10.0083 Da mass difference in a tryptic digested peptide, and the L-lysine-¹³C₆-¹⁵N₂ introduces +8.0142 Da mass difference respectively. The 1000x concentrated stock solutions with L-arginine-¹³C₆-¹⁵N₄ (88.8 mg/mL), L-lysine-¹³C₆-¹⁵N₂ (154 mg/mL) in PBS was filtered by 0.22-um syringe filter and stored at 4 °C. The pulse-SILAC medium was generated by adding the heavy-labeled amino acid stock into SILAC DMEM with 10% SILAC fetal calf serum. Cells were cultured in normal DMEM medium for 5–7 generation for pro-SILAC experiments. The culture medium was replaced with the pulse-SILAC medium for the indicated duration. For instance, when measuring MRF_4h of proteins, cells were incubated in pulse-SILAC medium for 3.5 h, and then the medium was replaced with pulse-SILAC medium containing 500 µM biotin-phenol probe for another 30 min. APEX labeling was triggered by adding 100x H₂O₂ solution (100 mM) into cell culture with gentle rocking. After 1-min labeling, the reaction medium was replaced with 500 µL quencher solution (1 mM sodium azide, 1 mM sodium ascorbate and 500 µM Trolox in PBS solution) for 2 min. Then the cells were washed again with quencher solution and twice with PBS.

For cell surface protein labeling, HEK293T-SS-HRP-KDEL cells were incubated in pulse-SILAC medium for 2 h. Then the medium was exchange to HBSS buffer containing 0.5 mg/mL sulfo-NHS-LC-biotin reagent for 2 min. The cells were wash with PBS buffer for three times

Cells were lysed with RIPA buffer containing 25 mM Tris•HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 2% SDS and protease inhibitors cocktail for 15 min at 4 °C. The high concentration of SDS facilitates the extraction of membrane proteins. Cells were scraped and lysed via ultrasonication on ice bath. The lysate was centrifuged at 20000 g at 4 °C for 10 min. For western blotting analysis, the collected supernatant was mixed with 5X loading buffer and heated at 95 °C for 10 min. For subsequent proteomic experiments, protein samples were purified by cold methanol precipitation at −80 °C for 3 h.