Agilent 6550 q tof

High-resolution MS spectra obtained on the LTQ-Orbitrap XL were processed using Xcalibur software (Thermo Fisher Scientific, USA). Lipid species were identified using an in-house database as well as online LipidMaps and Metlin lipid databases with an MS tolerance of 10 ppm. The identification was based on accurate mass and/or retention times (RT). Tandem MS data obtained on the Agilent 6550 Q-ToF were analyzed to obtain headgroup and/or fatty acid compositions and the RT for each lipid molecular species. For MRM data obtained on the Agilent 6460 QqQ, signal intensities were compared with the intensities from the spiked internal standards (PG 14:0 for PG and Lysyl-PG 16:0 for Lysyl-PG) and the retention times for the various classes were matched. Growth-phase MRM data were normalized to the dry cell pellet weights to ensure that comparisons were made per unit weight of bacterial sample. For the growth curve study, MRM peak areas for the early-logarithmic, mid-logarithmic and late-logarithmic phases were normalized to the peak areas for the late stationary phase to obtain a fold change. These fold changes were used to generate a heat map in R. The Student’s t test was performed to determine whether differences between OG1RF, Dap21 and Dap22 strains were statistically significant. p <0.05 was considered statistically significant.

Polar metabolite detection was performed on an Agilent 6550 Q‐TOF mass spectrometer operating in negative mode. Metabolites were separated on a SeQuant ZIC‐pHILIC column (5 µM, 150 × 4.6 mm, Millipore) using a binary gradient with a 1200 series HPLC system across a 45‐min method using 20 mM ammonium carbonate (pH 9) and acetonitrile as outlined in Cobbold etal, (2016). Two independent replicates of the metabolite profiling following AMR1 and Lipin depletion were performed using the same ZIC‐pHILIC chromatography on a Thermo Q‐Exactive operating in both positive and negative mode (rapid switching) as described previously (Creek etal, 2016). Lipid extracts were analysed on an Agilent 6550 Q‐TOF using the reverse phase chromatography outlined by Bird etal (2011).
GC‐MS analysis was performed using methods previously described (Saunders etal, 2011). Metabolites were separated using a BD5 capillary column (J&W Scientific, 30 m × 250 µM × 0.25 µM) on a Hewlett Packard 6890 system (5973 EI‐quadrupole MS detector). The oven temperature gradient was 70 °C (1 min); 70°C to 295°C at 12.5°C/min, 295°C to 320°C at 25°C/min; 320°C for 2 min. MS data were acquired using scan mode with a m/z range of 50–550, threshold 150 and scan rate of 2.91 scans/second. GC retention time and mass spectra were compared with authentic standards analysed in the same batch for metabolite identification.

The UV spectra were obtained by a Shimadzu UV-2600i spectrometer (Shimadzu, Kyoto, Japan). Optical rotations were measured by a JASCO P-2000 polarimeter (Anton Paar, Ostfildern, Germany). We recorded the HRESIMS data on Bruker microTOFQ-Q mass spectrometers (Billerica, MA, USA) and an Agilent 6550 Q-TOF (Agilent Technologies, Palo Alto, CA, USA). The NMR spectra were recorded on a Bruker AVANCE Ⅲ HD 600 MHz NMR spectrometer (Bruker BioSpin, Billerica, MA, USA) using TMS as an internal standard. The chromatographic silica gel (Qingdao Haiyang Chemical Factory, Qingdao, China), ODS (YMC Co., Ltd., Kyoto, Japan), and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden). We recorded the analytical HPLC data using a Shimadzu SPD-M20A series machine equipped with a YMC C-18 column (250 mm × 4.6 mm, 5 μm). We conducted the semipreparative HPLC using a Shimadzu LC-6AD series pumping system equipped with an SPD-20A UV detector and C-18 column (20 mm × 250 mm, 5 μm; YMC Co., Ltd.). All the organic solvents were purchased from Yuwang and Laibo Chemicals Industries, Ltd., Shenyang, China.

We used LC/MS analysis to identify the chemical profiling of PGWE. Chromatographic separation of the aliquots was performed by an Agilent 1290 infinity LC (Agilent Technologies, Palo Alto, United States) using an Agilent Eclipse Plus C18 column (2.1 × 50 mm, 1.8 µm) and a mobile phase composed of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The gradient program was as follows: 0–3 min, 5% B; 3–13 min, 5%–80% B; 13–15 min, 80% B; 15–17 min, 80%–5% B; and 17–20 min, 5% B. The flow rate was 0.3 mL/min, and the injection volume was 1 μL, which was injected into the column using a thermostatted HiP-ALS autosampler. Separated peaks were analyzed using an Agilent 6550 Q-TOF (Agilent Technologies), which provided high-resolution mass measurement. The instrument was equipped with a Jet Stream ESI source. The ESI spray voltage was set to 4000 V for positive ion mode and 3500 V for negative ion mode. Mass spectra were acquired in the 100–1000 m/z range.

After the preparation of the samples and fatty acid standards (described in the Supplementary Materials), chromatographic separation of the tumor tissues was performed with an Agilent 1290 Infinity LC (Agilent Technologies, Walbronn, Germany) using an Acquity UPLC BEH C8 column (50 × 2.1 mm, 1.7 µm particle size, Waters) at 40 °C. Gradient elution of solvent A was water–methanol (97:3, v/v) with 10 mM ammonium acetate and 15 mM acetic acid (pH 4) and solvent B was 100% methanol. The gradient program was set to the following: 5–50% B (0–5 min), 50–90% B (5–17 min), 90% B (17–20 min), and the column was then equilibrated with 5% B for 3 min at a flow rate of 0.3 mL/min. All samples and calibration standards (1 µL each) were injected into the column using a Thermostated HiP-ALS autosampler. The HPLC system was interfaced with the MS system, which was an Agilent 6550 Q-TOF (Agilent Technologies, Walbronn, Germany) equipped with a jet stream ESI source operating in negative ion mode. The ESI spray voltage was set to 3500 V (Vcap). Mass spectra were acquired at a scan rate of 1.0 spectra/s with a mass range of 100–1200 m/z. The procedure was controlled using Q-TOF Quantitative Analysis software (Version B 07.00, Agilent Technologies, Walbronn, Germany).