Fig. 2: Characterization of βES labeling in bacteria.

a Structures of L-methionine and its analog homopropargylglycine (HPG) used in BONCAT. b Structures of L-threonine and its analog β-ethynylserine (βES) used in THRONCAT. c Scheme of the BONCAT and THRONCAT workflows for NSP labeling in E. coli and their associated drawbacks and benefits. d–f, h In-gel visualization of βES or HPG incorporated into E. coli. Incorporated βES or HPG was conjugated to Cy5-azide using a copper-catalyzed azide-alkyne cycloaddition reaction and visualized through in-gel fluorescence scanning. Silver stain panels show total protein. Bar charts show the relative intensity of in-gel fluorescence normalized to silver stain intensity. The experiments were performed in biological triplicates with similar results. Representative gels are shown here. d Comparison of βES and HPG incorporation into E. coli BL21 lysate after 1 h incubation with 4 mM βES or 4 mM HPG in LB medium. A relative intensity bar chart is normalized to βES signal, which is set to 100% e Detection of βES and HPG in methionine auxotrophic E. coli B834 lysate after 1 h incubation with 4 mM βES or 4 mM HPG in methionine-free medium. Line scan of gel lanes shows differences in labeling intensities between corresponding bands in βES (blue line) and HPG (gray line) treated samples. Arrows indicate selected bands showing strong relative labeling in βES (blue arrows) and HPG (gray arrows) treated samples. Relative intensity bar chart is normalized to βES signal, which is set to 100%. Met, Methionine. f βES incorporation in E. coli BL21 over time. E. coli was incubated with 4 mM βES in LB medium for the indicated durations. Relative intensity bar chart is normalized to ‘βES 60 min’, which is set to 100%. g Growth curve of E. coli BL21 incubated with 1 mM or 4 mM βES. Bacterial growth was monitored by OD600 absorbance. Untreated E. coli (control) and E. coli treated with 34 μg/mL chloramphenicol were included as positive and negative controls, respectively. OD₆₀₀, optical density at 600 nm; AU, arbitrary units. Data are presented as mean values, error bars represent s.d., Sample size is n = 6. h βES detection in E. coli BL21 after 1 h incubation with 1 mM βES in LB medium. No βES incorporation is detected upon co-incubation with chloramphenicol (CAP) or a 50-fold excess of threonine. Relative intensity bar chart is normalized to ‘βES without CAP or Thr’ signal, which is set to 100%. CAP chloramphenicol, Thr L-threonine, kD Kilodalton. Source data are provided as a Source Data file.