Protein-based click chemistry

(1) The cross-linked cells were collected and 0.2% SDS was added (1× PBS) to extract protein. (2) Click chemistry was performed by adding cleavable azide–biotin reagents, THPTA, CuSO₄, and sodium ascorbate to the protein sample with the molar concentration ratio to the cross-linker of 1:10, 4:10, 0.5:10, and 1.25:10, respectively. The volume of the reaction was 2.5 ml. The resulting mixture was rotated at 60°C for 2 h (Supplementary Table S1). Then, the proteins were deposited by acetone precipitation. (3) The precipitated protein pellets were air dried and resuspended in 8 M urea (50 mM NH₄HCO₃), following by reduction (8 mM DTT, 25°C, 1 h) and alkylation (32 mM IAA, 25°C, 30 min, dark), and the samples were diluted to 1 M urea with 50 mM NH₄HCO₃ and digested with trypsin at 37°C overnight. The experimental procedure was consistent with our previous mature works (Gao et al., 2022; Zhao et al., 2022).