Acquity uplc beh c18 1.7 m column

Protein samples were rapidly thawed and injected onto an integrated fluidics system containing a HDX-3 PAL liquid handling robot and climate-controlled chromatography system (LEAP Technologies), a Dionex Ultimate 3000 UHPLC system, as well as an Impact HD QTOF Mass spectrometer (Bruker). Proteins were run over two immobilized pepsin columns (Applied Biosystems; Poroszyme Immobilized Pepsin Cartridge, 2.1 mm × 30 mm; Thermo-Fisher 2‐3131‐00; at 10 and 2°C, respectively) at 200 μL/min for 3 min. The resulting peptides were collected and desalted on a C18 trap column (Acquity UPLC BEH C18 1.7 µm column [2.1 × 5 mm]; Waters 186002350). The trap was subsequently eluted in line with a C18 reverse-phase separation column (Acquity 1.7 μm particle, 100 × 1 mm² C18 UPLC column, Waters 186002352), using a gradient of 5–36% B (buffer A 0.1% formic acid; buffer B 100% acetonitrile) over 16 min. Lipids were directly captured on the LC system, and eluted off at the 100% acetonitrile step, with no interference on mass spectrometer or LC performance. Full details of the LC setup and gradient for lipid samples are in Stariha et al., 2021 (link). Mass spectrometry experiments were performed on an Impact II QTOF (Bruker) acquiring over a mass range from 150 to 2200 m/z using an electrospray ionization source operated at a temperature of 200°C and a spray voltage of 4.5 kV.

Dried extracts were dissolved in methanol prior to the mass spectrometry (MS) analysis. MS experiments were carried out on a Dionex Ultimate 3000 UPLC system (ThermoFisher Scientific, Waltham, MA, USA) coupled to PDA detector (stationary phase 30 or 100 mm ACQUITY UPLC BEH C18 1.7 µm column (Waters Corporation, Milford, MA, USA), mobile phase: Linear gradient of [A] ddH₂O + 0.1% formic acid/[B] acetonitrile + 0.1% formic acid, 5% to 95% at flow rate of 0.6 mL/min). Further mass detection was performed coupling either an amaZon speed (Bruker, Billerica, MA, USA) or LTQ Orbitrap XL mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) using positive ionization mode and mass range detection of m/z 200 to 2000. Data analysis was performed using software Compass Data Analysis v. 4.1 (Bruker) and Xcalibur v. 3.0 (ThermoFisher Scientific).

Polyamines and amino acids were analyzed after derivatization with 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ-Tag Ultra Derivitization Kit, Waters, Mildorf, USA) according to the manufacturer’s instructions. Chromatographic separation was achieved using an Acquity UPLC BEH C18 1.7 µm column (2.1 x 50 mm, Waters, Mildorf, USA) with a pre-column. One microliter of sample was injected before running the solvent gradient: 0.01% B for 0.54 min, then up to 9.1% B in 6.5 min, 2 min at 21.2% B followed by 0.4 min at 59.6% B and then back to initial conditions in 0.6 min (total run time 10.1 min). The column was maintained at 55°C with a flow rate of 0.7 mL/min. The source temperature was set to 150°C and the desolvation temperature was set to 650°C. The capillary voltage was set to 3.0 kV. Nitrogen was used as the drying and nebulizing gas, with 600 L/h gas flow and 1200 L/h desolvation gas flow.

S. aurantiacus LU19075 was grown in 20 mL of TSB in a 100 mL baffled flask for one day, and 1 mL of the culture was inoculated into 100 mL of DNPM production medium in a 500 mL baffled flask. Cultures were grown for 6 days at 28 °C and 180 rpm in an Infors multitron shaker (Infors AG, Basel, Switzerland). Metabolites were extracted from the supernatant with butanol, concentrated to dryness, and dissolved in 1 mL methanol (MeOH). One microliter was separated on a Dionex Ultimate 3000 UPLC system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an ACQUITY UPLC BEH C18 1.7 µm column (30, 50, or 100 mm, Waters Corporation, Milford, MA, USA) using a linear gradient of 5–95 vol% aqueous acetonitrile (ACN) with 0.1% formic acid (FA) at a flow rate of 0.6 mL/min and a column oven temperature of 45 °C. Sample analysis was carried out on a coupled PDA detector followed by an amaZon speed (Bruker, Billerica, MA, USA) for production control. High-resolution masses were obtained from either an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) or a MaXis high-resolution LC-QTOF system (Bruker, Billerica, MA, USA) using positive ionization mode and mass range detection of m/z 200 to 2000. Data analysis was performed using Compass Data Analysis v. 4.1 (Bruker) and Xcalibur v. 3.0 (Thermo Fisher Scientific).

To detect DAF-FM-T, a theoretical product of NO and DAF-FM, in the DAF-FM DA-treated cells, an LC–MS/MS analysis was performed. The cell extracts were obtained as described above. Methanol was added to each cell extract to produce a 60% final concentration. The cell extracts were filtered and centrifuged at 12,000 g for 5 min to remove proteins, and the supernatant was used as the LC–MS/MS sample. LC–MS/MS analysis using an Acquity UPLC BEH C18 1.7-µm column (Waters, Milford, MA) was performed with an LCMS™-8045 (Shimadzu Corp., Kyoto, Japan). A gradient elution of 5–100% acetonitrile was used as the mobile phase, with a flow rate of 0.3 mL/min. The positive electrospray ionization mode was selected with fragments of m/z 413.0 > 369.0 (DAF-FM), 424.0 > 332.0 (DAF-FM-T), and 437.0 > 373.0 (UNK436). The standard DAF-FM-T was prepared in vitro as described previously using DAF-FM (Goryo Chemical Inc.) and a NO-donor^{38 (link)}.

Dry crude extracts were dissolved in methanol prior to MS analysis. Sample compounds were separated on a Dionex UltiMate 3000 UPLC (Thermo Fisher Scientific) coupled to a PDA detector (stationary phase: 100 mm ACQUITY UPLC BEH C18 1.7 µm column (Waters Corporation, Milford, MA, USA), mobile phase: linear gradient of [A] ddH₂O + 0.1% formic acid/[B] acetonitrile + 0.1% formic acid, 5% to 95% at flow rate 0.6 mL/min). Mass detection was carried out by coupling to an amaZon speed (Bruker, Billerica, MA, USA) or a MaXis II (Bruker) mass spectrometer, which provides a mass accuracy of 5 ppm. Spectra were aquired using positive ionization mode and with a mass selection range of m/z 100 to 2000. Compass Data Analysis 4.2 was used for mass spectrometry data analysis.