Machine-learning guided discovery of emergent antimicrobial activity from dynamic covalent assemblies（Escherichia coli，TMT protein quantitation）

In this dataset, Escherichia coli proteome responses were profiled by TMTpro-based quantitative proteomics across three treatment conditions, each with three biological replicates (total n = 9). The A5B5-treated samples are labeled as Ecoli.TMT_pro.A5B5_1, Ecoli.TMT_pro.A5B5_2, and Ecoli.TMT_pro.A5B5_3; the PBS-treated control samples are Ecoli.TMT_pro.PBS_1, Ecoli.TMT_pro.PBS_2, and Ecoli.TMT_pro.PBS_3; and the CHX-treated positive control samples are Ecoli.TMT_pro.CHX_1, Ecoli.TMT_pro.CHX_2, and Ecoli.TMT_pro.CHX_3. For TMTpro labeling, lyophilized peptide samples were resuspended in 100 mM TEAB (pH 8.5), and 40 μL of each sample was transferred for labeling; TMTpro reagent was dissolved in anhydrous acetonitrile, 10 μL reagent was added per sample, and reactions were incubated at room temperature for 1 h, then quenched with 5 μL of 5% hydroxylamine for 15 min. Labeled peptides were lyophilized and stored at −80°C before LC–MS/MS analysis. Mass spectrometry was performed on a Q Exactive HF (Thermo, USA) with a Nanospray Flex source, coupled to an EASY-nLC 1200 system, using a 25 cm × 75 μm C18 column at 300 nL/min over a 75 min gradient (0–40 min, 5–28% B; 40–60 min, 28–42% B; 60–65 min, 42–90% B; 65–75 min, 90% B), where mobile phase A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. Full MS scans were acquired over 350–1500 m/z at 60,000 resolution with AGC target 3e6, followed by top-20 HCD MS/MS (NCE 32) at 45,000 resolution with AGC target 2e5, maximum injection time 80 ms, dynamic exclusion 30 s, in positive ion mode. Raw files were searched using Proteome Discoverer v2.4 against the corresponding sample protein database with trypsin specificity; cysteine alkylation was set as a fixed modification, TMTpro labeling was used for quantification, global FDR was controlled at 0.01, and only protein groups supported by at least two peptides were retained for quantification.