General experimental procedures
Primer synthesis and DNA sequencing were performed by Genewiz. Purification of recombinant plasmids was performed using an EZNA Plasmid DNA Mini Kit from Omega Bio-Tek. Restriction enzymes were purchased from New England Biolabs and digestions were performed according to the manufacturer’s protocols. Gibson Master Mix was purchased from New England Biolabs and Gibson Assembly was performed according to the manufacturer’s protocol. Nickel nitriloacetic acid agarose (Ni-NTA) resin was purchased from Qiagen and Thermo Fisher Scientific. Benzylidenehydrazine was purchased from AstaTech. NADPH was purchased from Fisher Scientific. DBCO-acid was purchased from Conju-Probe. Diazoacetone was purchased from Enamine. Novex Tris-Glycine SDS–PAGE gels were purchased from Thermo Fisher Scientific. Protein concentrations were determined by measuring UV-absorption at 280 nm using a Thermo Scientific NanoDrop 2000. ExPASy ProtParam was used to calculate extinction coefficients. Optical densities of E. coli cultures were measured at 600 nm using a Beckman Coulter DU730 Life Sciences UV/Vis spectrophotometer. All water used was purified using a MilliQ water purification system. The remaining materials were purchased from Sigma Aldrich unless otherwise indicated. All experiments were performed in biological triplicates unless indicated and either individual data points with errors bars (± 1 s.d.) or representative results are shown. All starting materials used for chemical synthesis were either purchased from Sigma Aldrich or accessed synthetically as described below.
Sample preparation method 1
Ten microlitres of reaction mixture were diluted with 150 µl of LC–MS grade H 2 O, 20 µl of LC–MS grade acetonitrile (ACN), and 20 µl of LC–MS grade methanol and samples were filtered (0.2 µm, VWR, nylon).
Sample preparation method 2
Reaction mixtures were diluted with 70 µl of exchange buffer (20 mM HEPES pH 8.0, 10 mM MgCl 2 , 50 mM NaCl, 10% glycerol), 15 µl of LC–MS grade methanol, and 15 µl of LC–MS grade ACN and filtered (3 kDa, Pall, Omega membrane)
Agilent 6530 qTOF spectrometer with Dual AJS source
Unless otherwise indicated, samples were analysed by LC–MS using an Agilent 1200 series LC system coupled to an Agilent 6530 qTOF spectrometer with Dual AJS source. Drying gas temperature was 300 °C, drying gas flow was 11 l min−1, nebulizer pressure was 45 psi, sheath gas temperature was 275 °C, sheath gas flow was 11 l min−1, capillary voltage was 3,500 V, nozzle voltage was 500 V, fragmentor voltage was 125 V, and skimmer voltage was 65 V. Mass spectra were acquired in positive ion mode. A mass window of 10 ppm was used for EICs.
Thermo Orbitrap IQ-X Tribrid
Unless otherwise indicated, samples were analysed using a Horizon Vanquish UHPLC coupled to a Thermo Orbitrap IQ-X Tribrid mass spectrometer. The LC column was a Kinetex C18 column (1.7 µm, 100 Å, 150 × 2.1 mm, Kinetex). Two microlitres of sample were injected. The flow rate was 0.4 ml min−1 using mobile phase A (0.1% formic acid in water) and B (0.1% formic acid in ACN). The LC conditions were 0–2 min 10% B isocratic, 2–29 min 10–90% B, 29–32 min 90% B isocratic, 32–32 min 90–10% B, 32–35 min 10% B isocratic. The MS settings were: mass range 400–600 m/z, 120,000 resolution, the RF lens was 35%, the standard AGC target was used, and the auto maximum injection time mode was used. MS/MS spectra were acquired with a 1 m/z isolation window, 15,000 resolution, standard AGC target, auto maximum injection time mode and higher-energy collision-induced dissociation (HCD) fragmentation with 30% collision energy. Spectra were acquired in positive mode. A mass window of 5 ppm was used for EICs.
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