Acquity uplc 1 class plus

The chemical profile of the metabolites produced by TSA32-1 was analyzed using a Waters Acquity UPLC I-Class PLUS equipped with a Waters ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 µm) (Milford, MA, USA), maintained at an isothermal temperature of 65 °C. A binary pump delivered the mobile phase at the flow rate of 0.4 mL/min under gradient elution using two mobile phases, LC-MS–grade water + 0.1% v/v formic acid (solvent A) and LC-MS–grade acetonitrile + 0.1% v/v formic acid (solvent B). The auto-sampler was set to an injection volume of 1 µL. MS analysis was performed using a Waters Xevo-G2-XS QTOF LC-MS equipped with an electrospray ionization source. An analysis was conducted in positive-ion mode at a mass range of 100–1600 Da. The source temperature and capillary voltage were set to 150 °C and 3.0 kV, respectively. Ar was used as the collision gas. Standard solutions of surfactin (Santa Cruz Biotechnology, Dallas, TX, USA) and plipastatin (Sigma, St. Louis, MI, USA) were used.

An ACQUITY UPLC I-Class plus (Waters Corporation, Milford, CT, USA) coupled with a QE plus (Thermo Fisher Scientific, Shanghai, China) high-resolution tandem mass spectrometer was used to analyze the metabolic profiling in both ESI positive and ESI negative ion modes. An ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 × 100 mm) was employed in both positive and negative modes. The binary gradient elution system consisted of (A) water (containing 0.1% formic acid, v/v) and (B) acetonitrile (containing 0.1% formic acid, v/v) and separation was achieved using the following gradients: 0–2 min, 95% A, 5% B; 2–14 min, 95% to 0% A, 5% to 100% B; 14 min, 100% B; 15 min, 100% B; and 15.1–16 min, 95% A, 5% B. Formic acid and acetonitrile were provided by Thermo Fisher Scientific. The flow rate was 0.35 mL/min and the column temperature was 45 °C. All the samples were kept at 10 °C during the analysis. The injection volume was 3 μL.
The mass range was from m/z 100 to 1000. The resolution was set at 70,000 for the full MS scans and 17,500 for HCD MS/MS scans. The Collision energy was set at 10, 20 and 40 eV. The mass spectrometer operated as follows: spray voltage, 3800 V (+) and 3000 V (−); sheath gas flow rate, 35 arbitrary units; auxiliary gas flow rate, 8 arbitrary units; capillary temperature, 320 °C; aux gas heater temperature, 350 °C; s-lens RF level, 50.

In the three different laboratories, the instrument consisted of an ultra-high-performance liquid chromatography (UHPLC) system (Acquity UPLC I-Class Plus, Waters Milford, MA, USA) coupled to a time-of-flight (TOF) mass spectrometer (BioAccord Acquity RDa, Waters Milford, MA, USA). Each UHPLC system was equipped with a binary solvent delivery pump, a 50 µL mixer assembly (or a 380 µL mixer assembly in lab 2), a flow-through needle (FTN) sample manager, and a Tunable UV detector (TUV) operated in the single wavelength mode (λ = 214 nm, sampling rate 20 points/s). For all the experiments, the MS device was operated in the ESI+ mode with a capillary voltage of 1.5 kV, a desolvation temperature of 500 °C and a cone voltage of 30 V. The peptide fragmentation was obtained by ramping up the fragmentation cone voltage from 60 V to 130 V. The full scan acquisition was performed with Intelligent Data Capture (IDC) enabled and a mass range of 50–2000 m/z with a scan rate of 5 Hz. The data acquisition was performed with UNIFI Software (Waters, Milford, MA, USA): UNIFI v1.9.4 in lab 1, v1.9.9 in lab 2 and v1.9.13.9 in lab3.

Five micrograms (5 μg) of tryptic digested antibody were analysed on a BioAccord LC-MS system (Waters Corporation, United States). The system configuration includes an ACQUITY UPLC I-Class PLUS with an ACQUITY RDa detector (a compact time-of-flight mass detector) controlled by the compliance-ready UNIFI Scientific Information System software platform (Waters Corporation, United States). Samples were separated using an ACQUITY UPLC Peptide BEH C18 column (130 Å, 1.7 μm, 2.1 mm × 100 mm, Waters Corporation, United States) at 65°C and 250 μL/min, with a 60 min gradient from 1 to 40% of 0.1% formic acid in acetonitrile (mobile phase B). 0.1% formic acid in LCMS water was used as mobile phase A. The RDa mass detector was operated in positive electrospray ionisation full scan with fragmentation acquisition mode (MSe) at 2 Hz acquisition rate with a mass range of 400–2,000 m/z. The capillary voltage was set at 1.2 kV, cone voltage at 30 V, and the desolvation temperature was kept at 350°C. Leucine Enkephalin (Waters Corporation, United States) was used as the LockSpray compound for real-time mass correction.

Bacterial pellets were collected at 4 h after fresh culture, and shipped on dry ice. The metabolomic data analysis was performed by Shanghai Luming biological technology co., LTD (Shanghai, China). An ACQUITY UPLC I-Class plus (Waters Co., Milford, MA) fitted with Q-Exactive mass spectrometer equipped with heated electrospray ionization (ESI) source (Thermo Fisher Scientific, Waltham, MA) was used to analyze the metabolic profiling in both ESI positive and ESI negative ion modes. The original LC-MS data were processed by software Progenesis QI V2.3 (Nonlinear, Dynamics, Newcastle, UK) for baseline filtering, peak identification, integral, retention time correction, peak alignment, and normalization. A two-tailed Student's t-test was further used to verify whether the metabolites of difference between groups were significant. Differential metabolites were selected with VIP values greater than 1.0 and P-values less than 0.05.