Ultimate 3000 rslc

In vitro materials were ground with liquid nitrogen and mixed with 1 mL of methanol. Supernatant was collected by centrifugation (12000 rpm, 1 min). The LC-ESI-MS system consisted of an ultra-performance liquid chromatography system (Ultimate 3000 RSLC, Dionex) and an electrospray ionization source of quadrupole time-of-flight mass spectrometer (maXis HUR-QToF system, Bruker Daltonics). The autosampler was set at 4°C. Separation was performed with reversed-phase liquid chromatography on a BEH C8 column (2.1 × 100 mm, Walters). The elution started from 99% mobile phase A (0.1% formic acid in ultrapure water) and 1% mobile phase B (0.1% formic acid in ACN), held at 1% B for 1.5 min, raised to 60% B in 6 min, further raised to 90% in 0.5 min, and then lowered to 1% B in 0.5 min. The column was equilibrated by pumping 1% B for 4 min. The flow rate was set to 0.4 mL/min with an injection volume of 5 μL. LC-ESI-MS chromatogram were acquired under the following conditions: capillary voltage of 4500 V in positive ion mode, dry temperature of 190°C, dry gas flow maintained at 8 L/min, nebulizer gas at 1.4 bar, and acquisition range of m/z 100–1000.

The extraction was carried out by adding 40 μL of 100% DMF to 1 mg of the dry plant powder with vortexing for 30 min. After centrifugation at 16,000 × g for 10 min, the supernatant was collected for LC-ESI-MS analysis. The LC-ESI-MS system consisted of an ultra-performance liquid chromatography (UPLC) system (Ultimate 3000 RSLC, Dionex) and an electrospray ionization (ESI) source of quadrupole time-of-flight (TOF) mass spectrometer (maxis HUR-QToF system, Bruker Daltonics). The autosampler was set at 4°C. Separation was performed with reversed-phase liquid chromatography (RPLC) on a BEH C8 column (2.1 × 100 mm, Walters). The elution started from 50% mobile phase A (0.1% formic acid in deionized water) and 50% mobile phase B (0.1% formic acid in ACN), raised to 60% B in 2 min, further increased to 75% after an additional 1 min, and then raised to 100% B in 4.5 min, held at 100% B for 2.5 min, and then lowered to 50% B during the final 1 min. The column was equilibrated by pumping 50% B for 4 min. The flow rate was set 0.4 mL/min with an injection volume of 2 μL. LC-ESI-MS chromatogram was acquired in positive ion mode under following conditions: capillary voltage of 4500 V, dry temperature at 190°C, dry gas flow maintained at 8 L/min, nebulizer gas at 1.4 bar, and acquisition range of m/z 100–1000.

Enantioselective UHPLC analyses of CBC, isolated
as described above, were performed on an UltiMate 3000RSLC (Dionex,
Benelux, Amsterdam, The Netherlands). Specifically, the instrument
operation and chromatographic data acquisition and processing were
performed using the Chromeleon 7.2 chromatography data system. All
separations were performed using (R,R)-Whelk-O1 and (S,S)-Whelk-O1 CSPs,
prepared according to a previously described procedure starting from
Kromasil 1.8 μm silica particles and slurry packed into 100
× 4.6 mm (L × i.d.) stainless steel columns and commercially
available from Regis Technologies Inc. (Morton Grove, IL, USA). Isocratic
conditions were set as follows: mobile phase: n-hexane/isopropanol
(99.5:0.5 v/v); flow rate: 1.0 mL/min; T = 30 °C; detection: UV 280 nm, CD 280 nm.

Samples were separated using reversed-phase chromatography on a Dionex Ultimate 3000 RSLC nano-system. Using a flow rate of 30 µL/min, samples were desalted on a Thermo PepMap 100 C18 trap (0.3 × 5 mm, 5 µm) for 5 min, followed by separation on a Acclaim PepMap RSLC C18 (150 mm × 75 µm) column at a flow rate of 300 nL/min with a gradient of 10%–95% buffer B over 60 min where buffer A = 1% ACN/0.1% FA and buffer B = 80% ACN/0.1% FA. Eluted molecules were directly analysed on an Orbitap Elite mass spectrometer (Thermo, Brisbane, Australia) using an NSI electrospray interface. Source parameters included a capillary temperature of 275 °C; S-Lens RF level at 60%; source voltage of 2 kV and maximum injection times of 200 ms for MS. Data were deconvoluted using Protein Deconvolution software (Thermo).

Tryptic digestion of proteins was performed following the in-gel digestion method described by Shevchenko et al. [40 (link)]. The proteins were redissolved in 20 µL of 0.1% (v/v) trifluoroacetic acid (TFA) prior to Zip-tip clean up and identified in technical triplicates with a Thermo Scientific Q Exactive mass spectrometer connected to a Dionex Ultimate 3000 RSLC nano chromatography system. Proteins were separated on a C18 column (C18RP Reposil-Pur, 100 × 0.075 mm × 3 μm) over 60 min at a flow rate of 250 nL/min with a linear gradient of increasing acetonitrile from 1% to 27%. The mass spectrometer was operated in data-dependent mode; a high-resolution (70,000) MS scan (300–1600 m/z) was performed to select the twelve most intense ions and fragmented using high-energy C-trap dissociation for MS/MS analysis. Raw data from the Q-Exactive was processed using MaxQuant [41 (link),42 (link)] (version 1.6.3.4), incorporating the Andromeda search engine [43 (link)]. MS/MS spectra were matched against the E. coli O157:H7 NCTC12900 database. The sequence coverage threshold was set at ≥40%, and the number of unique peptides threshold was set at >5 for selection.

In 1 ml of solution containing 100 μM cysteine, 100 μM PVS, PVSN, or Bay 11-7082 in 20 mM NaHCO₃, pH 8.4, reaction was held at 37°C for 60 min. The resulting reaction products were subjected directly to the LC–ESI–MS analyses. The LC–ESI–MS system consisted of an ultra-performance liquid chromatography system (Ultimate 3000 RSLC, Dionex) and an electrospray ionization (ESI) source of quadrupole time-of-flight (TOF) mass spectrometer (maXis HUR-QToF system, BrukerDaltonics). The samples were kept in an autosampler at 4°C. Separation was performed with reversed-phase liquid chromatography (RPLC) on a BEH C₁₈ column (2.1 × 100 mm, Walters). The elution started from 99% mobile phase A (0.1% formic acid in ultrapure water) and 1% mobile phase B (0.1% formic acid in ACN), held at 1% B for 0.5 min, raised to 60% B in 6 min, further raised to 90% B in 0.5 min, held at 90% B for 1.5 min, and then lowered to 1% B in 0.5 min. The column was equilibrated by pumping 1% B for 4 min. The flow rate was set 0.4 ml/min with injection volume of 2 μl. LC–ESI–MS chromatogram were acquired under following conditions: capillary voltage of 4500 V in positive ion mode, dry temperature at 190°C dry gas flow maintained at 8 l/min, nebulizer gas at 1.4 bar, and acquisition range of m/z 100–1000.

BABA was quantified in material from plants treated with chemical inducers as well as after infection with PstDC3000. Plant material was harvested, flash-frozen and ground to fine powder in liquid nitrogen. The extraction protocol was conducted as described by Thevenet et al. (2017) (link); in brief 100 mg of ground tissue was extracted in 500 μL of 0.1% HCOOH/H₂O (v/v) containing the deuterium labeled internal standard (BABA-d₃) using a Retsch mixer mill. After centrifugation of the extract at 18400 g during 4 min, the supernatant was purified by solid phase extraction on an Isolute SCX-2 cartridge (1 mL, 100 mg). The eluate was concentrated to dryness in a centrifugal evaporator (Speedvac) at 35°C. Samples were finally resuspended in 300 μL (1: 3) organic mobile phase B/EtOH 80% (v/v) leading to a final concentration of internal standards of 50 ng mL⁻¹. Extracted BABA was quantified using an Ultimate 3000 RSLC (Dionex, Thermo Fisher Scientific) interfaced with a 4000 QTRAP (AB Sciex) by injecting 3.5 μL of extract on an Acquity UPLC BEH HILIC column (100 mm × 2.1 mm, 1.7 μm, Waters).