Sample analysis was carried out by MS-Omics (MSOmics, Vedbæk, Denmark, www.msomics.com ) as follows. Proteins were precipitated using methanol, and the metabolites were extracted using a liquid–liquid extraction using chloroform and water. The aqueous phase was dried under nitrogen flow. Samples were reconstituted in eluent A (ultrapure water with 10 mM ammonium formate + 0.1% formic acid). The analysis was carried out using a UPLC system (UPLC ACQUITY, Waters) coupled with a time of flight mass spectrometer (Xevo G2 ToF, Waters). An electrospray ionization interface was used. The analysis was performed in negative and positive ionization modes. The UPLC was performed using a slightly modified version of the protocol described previously [59 ]. Data-processing used MZmine 2 [60 (link)] followed by curation using a custom made an in-house protocol. Identification of compounds was performed using both peak retention times (compared against authentic standards included in the analytical sequence) and accurate mass (with an acceptable deviation of 0.005 Da).
Xevo g2 tof
Manufactured by Waters Corporation
Sourced in United States
The Xevo G2 ToF is a high-performance liquid chromatography-mass spectrometry (LC-MS) system designed for accurate mass measurement and reliable quantification. It features a time-of-flight (ToF) mass analyzer that provides high mass resolution and sensitivity for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Acquity uplc, by Waters Corporation (4 mentions)
Matlab, by MathWorks (2 mentions)
Matlab r2014b, by MathWorks (2 mentions)
Chemstation, by Agilent Technologies (2 mentions)
433a peptide synthesizer, by Thermo Fisher Scientific (1 mentions)
Massprep micro desalting column, by Waters Corporation (1 mentions)
Ultra performance liquid chromatography (uplc), by Waters Corporation (1 mentions)
Mannitol, by Merck Group (1 mentions)
Vacutainer, by BD (1 mentions)
Diaion hp 20, by Merck Group (1 mentions)
Silica gel 60, by Merck Group (1 mentions)
Jnm ecs400, by JEOL (1 mentions)
Acquity uplc 1 class system, by Waters Corporation (1 mentions)
Acquity uplc h class system, by Waters Corporation (1 mentions)
10 protocols using xevo g2 tof
1
Metabolomic Analysis of Biological Samples
2
Intracellular Metabolomics of BMDMs
3
Synthesis and Purification of Antimicrobial Peptides
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OAKs were synthesized in-house (433 A Peptide Synthesizer; Applied Biosystems, Foster City, CA, USA) by the solid-phase method using 9-fluorenylmethyloxycarbonyl (Fmoc) active-ester chemistry on 4-methylbenzhydrylamine (MBHA) resin. OAKs were then deprotected and cleaved from the resin using trifluoroacetic acid:H2O (95:5) and purified to >95% chromatographic homogeneity by reverse phase high performance liquid chromatography (RP-HPLC) using C18 column (Vydac), a flow rate of 2 ml/min and a linear acetonitrile gradient of 1%/min (Alliance; Waters, Milford, MA, USA). Peaks identity was verified by mass-spectrometry (Xevo G2 Tof; Waters, Milford, MA, USA). Purified OAKs were then lyophilized and kept as dry powder at −20 °C.
4
Characterizing Protein Cage Masses
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The subunit masses of AaLS-OT-2, AaLS-OT-1, and wt AaLS protein cages were analyzed using an electrospray ionization time-of-flight mass (ESI-TOF) mass spectrometer (Xevo G2 TOF, Waters, Milford, MA, USA) interfaced to a Waters UPLC and an autosampler. Samples were loaded onto the MassPREP Micro desalting column (Waters) and eluted with a gradient of 5-95% (v/v) acetonitrile containing 0.1% formic acid with a flow rate of 300 µL/min [11 ]. Mass spectra were acquired in the range of m/z 500-3,000 and processed using MaxEnt 1 and MaxEnt 3 from MassLynx version 4.1 to obtain the average mass from multiple charge state distributions.
5
Intestinal Permeability Assessment in Pigs
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Intestinal permeability was assessed in vivo on d 28 and 29 after weaning. Eight pigs per treatment (representative of the median BW of each treatment) were fasted for 2 h and subsequently sedated with a mixture of xylazine (1.5 mg/kg BW) and ketamine (11 mg/kg BW) administered intramuscularly (i.m.) in order to minimize handling stress. After 10 min, animals were intragastrically dosed (gastroduodenal feeding tube, Levin type; VEC Medical) with a marker solution containing 0.15 g mannitol/kg BW (Sigma-Aldrich, Madrid, Spain) and 0.1 g Co-EDTA/kg BW [27 (link)] dissolved in 15 mL deionized water and immediately after were challenged with LPS as previously described. Blood samples were collected by jugular venipuncture 1 h after oral infusion of permeability markers into 5 mL evacuated fluoride/K-oxalate glucose blood collection tubes (BD vacutainer, Madrid, Spain). Plasma mannitol was determined by ultra-high performance liquid-chromatography mass-spectrometry (Xevo G2 TOF, Waters) as previously described [28 (link)]. The plasma concentration of cobalt was determined by atomic absorption spectroscopy [27 (link)].
6
Purification and Identification of Bioactive Compound
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The cellular Pol stimulated compound was purified from the screened medicinal plant using various column chromatography, consisting of Diaion HP-20 (Sigma Aldrich, St. Louis, MO, USA) and silica gel 60 (Merck Millipore, Darmstadt, Germany), and HPLC, consisting of Chromatorex ODS DM1020T (Fuji Silysia Ltd., Durham, NC, USA). The purified compound was identified using high-resolution mass spectrometry (Xevo G2 Tof: Waters; Milford, MA, USA) and nuclear magnetic resonance equipment (JNM-ECS400: JEOL RESONANCE; Tokyo, Japan).
7
Mass Spectrometry-Based Metabolomics Analysis
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Sample analysis was carried out by MS-Omics as follows. Intracellular extracts were dried under nitrogen flow and reconstituted in a MilliQ water/acetonitrile (1:1) mixture. Extracellular supernatants were diluted 1:3 in MilliQ water before acetonitrile was added (1:1). The analysis was carried out using a UPLC system (UPLC Acquity, Waters) coupled with a time of flight mass spectrometer (Xevo G2 Tof, Waters). An electrospray ionization interface was used as the ionization source. Analysis was performed in negative and positive ionization mode. The UPLC was performed using a slightly modified version of the protocol described by Paglia et al.47 (link) Data processing was carried out using MZmine 248 (link) followed by curation using a custom made in-house protocol. The compounds were identified using both peak retention times (compared with authentic standards) and accurate mass (with an accepted deviation of 0.005 Da). Metabolomic data were analyzed using MetaboAnalyst software (www.metaboanalyst.ca ).
8
Metabolite Profiling by UPLC-MS
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UPLC analyses were performed on a Waters Acquity UPLC I-class system equipped with a photodiode array detector. All analyses were performed using a reversed-phase UPLC column (Aeris Widepore 3.6 μm XB-C18 150 × 2.10 mm, purchased from Phenomenex). Ultraviolet absorbance was monitored at 254 nm. Column temperature was kept at 35 °C. UPLC-MS was performed using a Waters Acquity UPLC H-class system coupled to a Waters Xevo-G2 TOF. The mass spectrometer (MS) was operated in the positive electrospray ionization mode. Injection volume was 5 μl of a 3.8 mM library subjected to a 1:50 dilution in a solution of 0.6 v% of trifluoroacetic acid in doubly distilled water. Eluent flow was 0.3 ml min−1; eluent A: UPLC grade water (0.1 v% trifluoroacetic acid); eluent B: UPLC grade acetonitrile (0.1 v% trifluoroacetic acid). For full method, see Supplementary Table 3 . For UPLC-MS compound identification see Supplementary Table 4 .
9
Intracellular Metabolomics of BMDMs
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Metabolite extraction of BMDMs was performed on 2.5 million cells per well using 70°C aqueous 70% ethanol as described previously37 (link). Briefly, [13C]-Glutamine (1mM) was added on free-glutamine media cells for 4 hours. At collection, cells were placed immediately on ice, the media was removed, and cells were washed three times with ice-cold PBS to remove residual media. Intracellular metabolites were extracted twice with hot ethanol. For LCMS, samples where dried under nitrogen flow and reconstituted in a milliQ water/acetonitrile (1:1) mixture for injection using a UPLC Acquity (Waters) separation system coupled with a Xevo G2 ToF (Waters). Compounds were ionized using an electrospray ionization source in negative mode. Data processing was performed in MATLAB (Mathworks, Inc.) using a custom-made in-house protocol. Compound identification was performed using both retention time of authentic standards and accurate mass with an accepted deviation of 0.005 Da. Raw data was converted to netCDF format using Chemstation (Agilent), before processing in MATLAB R2014b (Mathworks, Inc.) using PARADISe software. All MS sample processing and analysis were performed by MS-Omics, Inc. (Copenhagen, Denmark).
10
Mass Spectrometry Characterization Protocol
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Analyses were carried out on a Waters XEVO® G2 ToF. ESI source conditions: capillary voltage: 0.7 kV, cone voltage: 30 V, extraction cone: 4V, source temperature: 120°C, desolvation temperature: 400°C, gas flow: 10L/hr, desolvation gas flow: 1200L/hr. The analyses were carried out in positive and negative modes. The mass axis was calibrated with sodium formate in the range 50 to 1200 Da. Mass Lockspray: Leucine Enkephalin was used to correct masses. Two masses were checked in each mode (positive mode: 278.1141; 556.2771; negative mode: 236.1035; 554.2615) . The acquisitions were carried out in MS E mode using argon as collision gas to obtain analyte fragmentation. MS E mode enables to acquire simultaneously, mass spectra at both low and high collision energy, the former to obtain parent ion, the latter to produce fragment ions.
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