Liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis of the cross-coupling reactions was conducted on a Thermo Scientific Dionex Ultimate 3000 Rapid Separation LC system using XBridge BEH C18 column (130 Å, 3.5 µm, 2.1 × 100 mm). The flow rate was set to 0.35 ml min−1 and the column temperature was maintained at 40 °C. A generic binary gradient elution was carried out using different ratios of eluents A (water containing 0.1% formic acid) and B (acetonitrile). Following gradient was used: 0–0.5 min (5% B), 0.5–9.5 min (5% to 95% B), 9.5–11.5 min (95% B), 11.5–12.0 min (95% to 5% B) and 12.0–15.0 min (5% B). The mass spectrometric analysis was performed on an Orbitrap Velos Pro™ mass spectrometer system equipped with a Thermo Scientific Ion MAX API source housing. The MS conditions were as follows: heated electrospray ionization (HESI-II) probe, positive ionization mode, spray voltage 3.5 kV, capillary temperature 350 °C, normalized collision energy 35% for collision induced dissociation, and sheath gas and auxiliary gas flow rates of 35 and 10 arbitrary units, respectively. Full scan MS spectra (from m/z 100–1000) were acquired in the orbitrap with resolution R = 60 K.
Dionex ultimate 3000 rapid separation lc system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Dionex Ultimate 3000 Rapid Separation LC system is a high-performance liquid chromatography (HPLC) instrument designed for rapid and efficient separation of analytes. The system features a modular design and can accommodate a variety of HPLC column sizes and detection methods.
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Lab products found in correlation
Xcalibur 2 2 sp1, by Thermo Fisher Scientific (2 mentions)
Chromeleon 7, by Thermo Fisher Scientific (2 mentions)
Ion max api, by Thermo Fisher Scientific (1 mentions)
Hpx 87p column, by Aminex (1 mentions)
Refractomax 521, by Thermo Fisher Scientific (1 mentions)
Luna c18, by Phenomenex (1 mentions)
Ltq orbitrap velos, by Thermo Fisher Scientific (1 mentions)
Trypsin gold, by Promega (1 mentions)
Avance 400 nmr spectrometer, by Bruker (1 mentions)
5 mm bbfo probe, by Bruker (1 mentions)
Acclaim rslc 120 c18 column, by Thermo Fisher Scientific (1 mentions)
Topspin 3, by Bruker (1 mentions)
8 protocols using dionex ultimate 3000 rapid separation lc system
1
LC-HRMS Analysis of Cross-Coupling Reactions
2
HPLC Analysis of UDP-GlcA in Yeast
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UDP-GlcA produced during the standard and coupled HPLC enzyme assays as well as UDP-GlcA produced in metabolic pathway design experiments in S. cerevisiae cells were measured by a Dionex UltiMate 3000 Rapid Separation LC System (Thermo Scientific) using a NUCLEOSIL® 4000-7PEI (Macherey-Nagel) strong anion exchange (SAX) column cartridge (125×4.0 mm column size) for chromatography of nucleotides analyzing data with Chromeleon 7.12 (Thermo Scientific). Temperature of the column compartment was set at 25°C during analysis and separation of the different assay components was performed using the following eluents: A (2.5 mM Tris/phosphate (pH 7.2) and B (2.5 mM Tris/phosphate (pH 8.0)+1.5 M KCl), and the following gradient for standard and coupled HPLC enzyme assays: 5% B to 95% B in 5 min, flow rate 1.3 ml·min−1. Elution times of reference components were 2.06 min for UMP, 2.44 min for AMP, 2.87 min for UDP-GlcA, 3.85 min for UDP, 4.54 min for ADP, 5.28 min for UTP and 6.09 min for ATP. Separation of NDP-sugars from S. cerevisiae was performed using the following gradient: 5% B to 19.9% B in 5.8 min, 19.9% B to 21.9% B in 3.2 min and 21.9% B to 95% B in 1 min, flow rate 1.3 ml·min−1. Elution time of reference component was 5.96 min for UDP-GlcA. UV spectra were recorded between of 240 nm and 300 nm and the chromatogram was displayed for 260 nm.
3
Glucose Quantification via HPLC
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Glucose concentration was determined using High Performance Liquid Chromatography with an Aminex HPX-87P column. Supernatant from 1 mL of sample culture as filtered with a 0.2 μm Nylon filter before analysis using a Thermo Scientific Dionex Ultimate 3000 Rapid Separation LC system with RefractoMax 521 detector. Filtered and degassed water was used as the mobile phase with a flow rate of 0.6 mL/min. Column temperature was maintained at 85 °C. As necessary, supernatant samples were diluted with deionized water before filtration to stay within the linear range of the detector.
4
Phytochemical Analysis of Leaf Extracts
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The chemical portioning of the leaf ethanolic extracts was performed by High Performance Liquid Chromatography (HPLC) coupled both with a Diode Array Detector (DAD) and Mass spectrometer (MS). The analysis was conducted using Dionex UltiMate®3000 Rapid Separation LC system (Thermo Fischer Scientific, San Jose, CA, USA), with an auto-sampler controlled by Chromeleon 7.2 Software (Thermo Fisher, Bremen, DE and Dionex Softron GmbH, Germering, DE). The column was a Phenomenex Luna C18 (3.00 μm, 150 mm × 3.0 mm i.d., Phenomenex, Torrance, CA, USA), packed with core-shell particles of 2.5 μm. The flow rate was set at 200 μL·min−1 at 25 °C.
5
Proteomic Analysis by Mass Spectrometry
6
Lipidomics Analysis of Adipose Tissue
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The sample preparation and the lipidomics analysis were undertaken as described previously [44 (link)]. In brief, eWAT or cells (20 mg) were homogenized with ultrapure water (200 μl) and then extracted with chloroform-methanol (2:1) solution (1000 μl). The samples were incubated at 37 °C for 30 min and subsequently centrifuged at 16,000 g for 20 min at 4 °C. The lower organic phase (approximately 500 μl) was collected and evaporated. The organic residue was dissolved in isopropanol-acetonitrile (1:1) solution (100 μl). Samples were analyzed by the Thermo Scientific Dionex UltiMate 3000 Rapid Separation LC system (Thermo Fisher Scientific, Waltham, MA, USA). Peak extraction and integration were operated with Xcalibur 2.2 SP1.48 software (Thermo Fisher Scientific, Waltham, MA, USA).
7
Lipidomics Analysis of Adipose Tissue
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The serum and adipose sample preparations and the lipidomics analyses were undertaken as described previously (Jiang et al., 2015 (link)). In brief, epididymal white adipose tissue (eWAT) (20 mg) were homogenized with ultrapure water (200 μl) and then extracted with chloroform-methanol (2:1) solution (1,000 μl). The samples were incubated at 37°C for 30 min and subsequently centrifuged at 16,000 g for 20 min at 4°C. The lower organic phase (approximately 500 μl) was collected and evaporated. The organic residue was dissolved in isopropanol-acetonitrile (1:1) solution (100 μl). Samples were analyzed using the Thermo Scientific Dionex UltiMate 3000 Rapid Separation LC system (Thermo Fisher Scientific, Waltham, MA, United States). Peak extraction and integration were performed using Xcalibur 2.2 SP1.48 software (Thermo Fisher Scientific, Waltham, MA, United States).
8
Characterization of 17-Hydroxygeranyllinalool
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17-Hydroxygeranyllinalool was provided by HPC24 Standards (www.hpcstandards.com) and the general structure was verified by 1D and 2D NMR spectroscopy (for 1 H NMR, see Supplemental Figure 12). A Bruker AVANCE 400 NMR spectrometer (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted August 27, 2020. ; https://doi.org/10.1101/2020.08.26.267690 doi: bioRxiv preprint (Bruker, Rheinstetten, Germany), equipped with a 5 mm BBFO probe, was used to record 1 H NMR, DEPT 135, 1 H-1 H COSY, HSQC and HMBC spectra in MeOH-d 4 at 300 K. Spectra were processed using TOPSPIN 3.1. (BrukerBiospin). Homogenized samples were then centrifuged at 16 000 × g for 20 min at 4°C. The supernatant was centrifuged again at 16 000 × g for 20 min at 4°C. Two independent chromatographic methods were used to resolve HGL-DTGs. Both methods used a mixture of solvent A: water with 0.1% acetonitrile and 0.05% formic acid and solvent B: acetonitrile and 0.05% formic acid. U(H)PLC for method A was performed using a Dionex UltiMate 3000 rapid separation LC system (Thermo Fisher, http://www.thermofisher.com), combined with a Thermo Acclaim RSLC 120 C18 column (particle size 2.2 µm, average pore diameter 120Å, column dimension 2.1 × 150 mm).
The copyright holder for this preprint this version posted August 27, 2020. ; https://doi.org/10.1101/2020.08.26.267690 doi: bioRxiv preprint (Bruker, Rheinstetten, Germany), equipped with a 5 mm BBFO probe, was used to record 1 H NMR, DEPT 135, 1 H-1 H COSY, HSQC and HMBC spectra in MeOH-d 4 at 300 K. Spectra were processed using TOPSPIN 3.1. (BrukerBiospin). Homogenized samples were then centrifuged at 16 000 × g for 20 min at 4°C. The supernatant was centrifuged again at 16 000 × g for 20 min at 4°C. Two independent chromatographic methods were used to resolve HGL-DTGs. Both methods used a mixture of solvent A: water with 0.1% acetonitrile and 0.05% formic acid and solvent B: acetonitrile and 0.05% formic acid. U(H)PLC for method A was performed using a Dionex UltiMate 3000 rapid separation LC system (Thermo Fisher, http://www.thermofisher.com), combined with a Thermo Acclaim RSLC 120 C18 column (particle size 2.2 µm, average pore diameter 120Å, column dimension 2.1 × 150 mm).
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