Extended Data Fig. 6: Investigating the proteome reactivity of N-sulfonyl oxaziridine probe.

a) Oxidative cyclization bioconjugation by Trp-CLiC was tryptophan-selective in cell lysate models. Cell lysate (1 mg/mL) was labelled with Ox-W18 (1 mM), and then clicked with azide-dye. Fluorescence imaging showed that Trp-CLiC also works for cell proteomes. Excess free Ac-Trp-OMe could significantly inhibit the Trp-CLiC labelling, indicating the chemoselectivity of our method. Results are representative of three biological replicates. b) Dose-dependent labelling of cell lysate indicated that 250 μM Ox-W18 was enough for the labelling of proteome (1 mg/mL). Results are representative of two biological replicates. c) The stability of biotin-labelled proteome was tested in 50 mM Tris buffer or PBS (pH 8), showing that Tris buffer indeed greatly enhances cycloadduct stability. Results are representative of three biological replicates. d-e) CID-cleavage mechanisms for Trp-CLiC labelling and representative spectra. d) Proposed CID-cleavage mechanisms of the acid-cleavable biotin probe cycloadduct product. The unique reporter ions 213.17 and 425.16 could be utilized to further confirm tryptophan bioconjugation by Trp-CLiC. e) Proposed CID-cleavage mechanisms of the desthiobiotin cycloadduct product. The reporter ion is m/z 484.32. The signal peaks marked with asterisks represent the peptide products after MS cleavage and neutral loss.