6130 single quadrupole mass spectrometer

Human Ub DNA was subcloned into a pET60 vector (Novagen, 71851) for ubiquitin-6His-tag. PCR mutagenesis was used to generate Ub[TVLN]-6His-tag mutant. The corresponding proteins were produced in Rossetta (DE3) E. coli cells grown in 1 L terrific broth (Sigma-Aldrich, T0918). When the OD reached 0.6, the proteins were induced using 1 mM IPTG (Goldbio, I2481C) and further incubated for 21 h at 25°C. The E. coli cells were then collected, resuspended in 40 ml lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.06% BME, 2 tablets of Roche protease inhibitors), and lysed by tip-sonication. After removing the cell debris through ultracentrifugation (20,000 x g, 10 min), the supernatant was filtered through a 0.2-µm filter and incubated with the Ni-NTA beads (Pierce, 88221). After three times of wash, the 6XHis tagged proteins were eluted with Tris-HCl buffer containing 500 mM imidazole. The eluate was then loaded on a gel-filtration Superose 6 10/300 GL (GE Healthcare, 17517201) for further purification. Purity and integrity of the proteins were confirmed by Coomassie Brilliant Blue staining and intact mass analysis using Agilent 6130 Single Quadrupole Mass Spectrometer with 1260 LCMS (Expected MS for Ub^WT-6xHis (max isotopic m/z) = 9387.99127, measured [M + H] = 9388.03; Expected MS for Ub^TVLN-6xHis (max isotopic m/z) = 9386.97084, measured [M + H] = 9387.03).

³¹P-NMR spectra were acquired on Unity INOVA 400 spectrometer (161.84 MHz for ³¹P and 399.882.54 MHz for ¹H) (Varian, Palo Alto, CA, USA) equipped with a variable temperature controller and a Varian 5 mm Autoswitchable probe with Z-axis gradient optimized for tuning of ³¹P and ¹H [13 (link),14 (link),15 (link)]. The ³¹P chemical shifts are reported using an external reference standard (neat solution of phosphoric acid at room temperature, ~25 °C, δ ppm = 0.0). A ³¹P 45° flip angle pulse was used for both proton decoupled and non-decoupled spectra (90° ³¹P-pulse of 9.8 μs at 54 dB attenuation, where max power output is ~300 W). For proton decoupled spectra a composite pulse Waltz decoupling sequence was applied with field strength of 2525 Hz during the acquisition time of 1 s and the relaxation time of 1 s. Samples were dissolved in D₂O and the signal was averaged from 512 transients. Mass spectrometry was performed in negative ion mode on a 6130 Single Quadrupole Mass Spectrometer (Agilent, Santa Clara, CA, USA) attached to an Agilent 1200 HPLC.

LC-MS data were collected on an Agilent 1260 HPLC coupled to an Agilent 6130 single quadrupole mass spectrometer and an inline Agilent UV absorbance detector (210 nm) using 2.0-kV electrospray ionization (ESI) capillary voltage. Path length was 0.6 cm. Then, 100 mM samples (referring to the original amino acid and hydroxy acid concentration) were separated via a Kinetex XB-C18 column (150 × 2.1 mm, 2.6 μm particle size) under the same conditions described in the HPLC method. Eluted peaks were detected in negative-mode ESI-MS, scanning from 65–2000 m/z with 70 V source fragmentation voltage.

Compounds were analysed at 210 nm using an Agilent Ecilpse XDB-C18 column (5 μm, 4.6 mm × 250 mm) on an Agilent 1200 HPLC coupled with an Agilent 6130 single quadrupole mass spectrometer. High-resolution mass spectrum of FX1 was collected on an Agilent 6210 LCMS. Four HPLC programmes were used. Programme 1 (for Figs 1c and 3c,d and Figs 4b,d,f and 6): 5–90% acetonitrile–water with 0.1% formic acid from 0 to 30 min, 90–100% acetonitrile–water with 0.1% formic acid from 30 to 35 min and 100% acetonitrile–water with 0.1% formic acid from 35 to 50 min at 1 ml min⁻¹. Programme 2 (for Figs 2b–d and 3e and Figs 4a,c,e and 5a,c): 80–100% acetonitrile–water with 0.1% formic acid over 20 min at a flow rate of 1 ml min⁻¹. Programme 3 (for Fig. 5d): 50–100% acetonitrile–water with 0.1% formic acid over 30 min at 1 ml min⁻¹. Programme 4 (for Fig. 3b): 5–70% acetonitrile–water with 0.1% formic acid over 40 min at 1 ml min⁻¹. Compound purification was performed on the same HPLC.

The NRPSs were expressed in S. cerevisiae BJ5464-NpgA. The correct transformants were selected by autotrophy of uracil and/or tryptophan. Yeast strains harbouring one plasmid were cultured in 50 ml of SC-Ura (or -Trp) dropout medium (6.7 mg l⁻¹ yeast nitrogen base; 20 mg l⁻¹ glucose; 0.77 mg l⁻¹ -Ura or 0.74 mg l⁻¹ -Trp dropout supplement) at 30 °C with shaking at 250 rpm. For co-expression experiments, -Trp/-Ura dropout was used. After the OD₆₀₀ value reached 0.6, an equal volume of YP medium (10 mg l⁻¹ yeast extract; 20 mg l⁻¹ peptone) was added, and the cultures were maintained under the same conditions for an additional 3 days. The cultures were then extracted three times with 100 ml of ethyl acetate and subjected to analysis on an Agilent 1200 HPLC (at 210 nm) coupled with an Agilent 6130 single quadrupole mass spectrometer. The product titres were calculated from three independent experiments based on the standard curves of purified compounds.

The specificity of the several SpeG enzymes (SaSpeG from Staphylococcus aureus, BtSpeG from Bacilus thuringiensis, EcSpeG from Escherichia coli, and VcSpeG from Vibrio cholerae) for N¹ vs. N⁸ amines of spermidine was assessed using the following procedure. All proteins were expressed and purified as described previously and above [26 (link),29 (link),30 (link)]. The cleaved SpeG enzymes (9 ng of SaSpeG, 15 ng of BtSpeG, 25 ng of EcSpeG, and 25 ng of VcSpeG) were reacted with 2 mM spermidine and 1 mM AcCoA in 70 mM Bicine pH 8.0, 20 mM NaCl, and 0.005% Triton X-100 in a total reaction volume of 100 μL for 5 min at 22 °C. The reactions were stopped with 25 μL of 1 M guanidine HCl, and then reaction products were analyzed as described previously [29 (link)]. Briefly, polyamines were derivatized using dansyl chloride and were analyzed using a 1290 UPLC system equipped with a 6130 single quadrupole mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Standards included N¹ and N⁸-acetylspermidine dihydrochloride (Millipore Sigma Cat# 01467 and Santa Cruz Biotechnology, Dallas, TX, USA Cat# SC-236151A, respectively) and spermidine trihydrochloride (Millipore Sigma, Burlington, MA, USA, Cat#85578). Data reported were consistent across two technical replicates.