Photolabeled and PBS-washed E. coli were lysed in 50 mM ammonium bicarbonate (AmBic) containing protease inhibitor cocktail (catalog no. 11704900, Roche) and 0.1% RapiGest (catalog no. 186002122, Waters) by six intervals of 30-s ultrasound sonication in a vial tweeter (Hielscher Ultrasonics GmbH) at a power of 170 W and 80% cycle time. Protein concentration was determined by a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific Inc.), and 10 mg of protein was incubated with 200 μl of streptavidin agarose resin (catalog no. 53116, Thermo Fisher Scientific) for 100 min at 4°C to bind biotinylated proteins. Beads were settled by centrifugation at 2000g for 5 min and transferred to Mobicol columns equipped with a 30-μm pore size filter (MoBiTec GmbH). Beads were extensively washed with 5 M NaCl, StimLys buffer [50 mM tris (pH 7.8), 137 mM NaCl, 150 mM glycerol, 0.5 mM EDTA, 0.1% Triton X-100], 100 mM NaHCO3, and AmBic to remove nonbiotinylated proteins. Bead-bound proteins were reduced with 5 mM tris(2-carboxyethyl)phosphine in AmBic for 40 min at 37°C, alkylated with 10 mM iodoacetamide in AmBic for 30 min at 37°C, and proteolytically digested by sequencing-grade modified trypsin (catalog no. V511A, Promega) at an enzyme-to-protein ratio of 1:100 for 20 hours at 37°C. Released peptides were acidified and subjected to C18 purification using UltraMicroSpin Columns (The Nest Group).

Peptide samples were separated by reversed-phase chromatography on an HPLC column (75-μm inner diameter, New Objective) that was packed in-house with a 15-cm stationary phase (ReproSil-Pur C18-AQ, 1.9 μm) and connected to a nanoflow HPLC with an autosampler (EASY-nLC 1000, Thermo Fisher Scientific). The HPLC was coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) equipped with a nanoelectrospray ion source (Thermo Fisher Scientific). Peptides were loaded onto the column with 100% buffer A [99.9% H2O, 0.1% formic acid (FA)] and eluted at a constant flow rate of 300 nl/min with a 30-min linear gradient from 6 to 20% buffer B (99.9% MeCN and 0.1% FA) followed by a 15-min transition from 20 to 32% buffer B. Electrospray voltage was set to 2 kV, sheath and auxiliary gas flow to zero, and capillary temperature to 250°C. In data-dependent acquisition (DDA) mode, the mass spectrometer automatically switched between precursor and fragment ion detection. Following a high-resolution survey mass spectrum [from 300 to 1500 mass/charge ratio (m/z)] acquired in the Orbitrap with resolution R = 70,000 at m/z 200 (automatic gain control target value 3 × 106), the 15 most abundant peptide ions with a minimum intensity of 2.5 × 104 were selected for subsequent higher-energy collisional dissociation fragmentation with an isolation window of 1.4 Da, and fragments were detected by MS/MS acquisition in the Orbitrap at resolution R = 35,000 (automatic gain control target value of 1 × 106). Target ions already selected for fragmentation were dynamically excluded for 30 s.

Acquired raw files were subjected to protein identification using Comet (v.2015.01) and Trans Proteomic Pipeline v.4.7 (Seattle Proteome Center/Institute of Systems Biology, Seattle) by matching ion spectra acquired in the DDA mode against a SwissProt (UniProt consortium)–reviewed E. coli protein database (downloaded November 2016). Peptides were required to be fully tryptic with a maximum of two missed cleavage sites, carbamidomethylation as a fixed modification and methionine oxidation as a dynamic modification. The precursor and fragment mass tolerance were set to 20 parts per million (ppm) and 0.02 Da, respectively. Identified proteins were quantified by integration of chromatographic traces of peptides using Progenesis QI v.2.0 (Nonlinear Dynamics, UK). Contaminant hits were removed, and proteins were filtered to obtain an FDR of <1%. Raw protein abundances based on nonconflicting peptides were exported, and differential abundance testing was performed using R package MSstats v3.5.3 (33). Significantly enriched E. coli proteins (abundance fold change ≥ 2 and adjusted P values of ≤0.05) were considered as bona fide thanatin-PAL5–binding candidates. Mass spectrometric data were deposited to the ProteomeXchange Consortium (www.proteomexchange.org/) via the PRIDE partner repository (data set identifier: PXD010988).

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