Measurements of 12C/13C-toxin- and mock-treated samples were performed with an UltiMate 3000 HPLC system combined with an Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Chromatographic settings were as follows: column Kinetex C18 (150 × 2.1 mm, 2.6 μm; Phenomenex, Aschaffenburg, Germany); column temperature 25 °C; eluents 0.1% formic acid and 5 mM ammonium formate in water (eluent A) and in methanol (eluent B); flow rate 250 μL/min. Injection volume was set to 10 μL and Gradient method 1 (30 min gradient, from 10% to 100% B plus re-equilibration) was used as described in [32 (link)]. Orbitrap measurements were performed in positive and negative electrospray ionisation mode separately with a scan range from m/z 130 to 1300. All other mass spectrometric settings were concordant with those of Kluger et al. [25 (link)]. Data were acquired and evaluated with Thermo Xcalibur 4.0.27.10 and 2.2 software (both Thermo Fisher Scientific), respectively.
Exactive plus orbitrap mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Exactive Plus Orbitrap mass spectrometer is a high-performance instrument that utilizes Orbitrap technology to provide accurate mass measurements. It is designed to deliver robust and reliable performance for a variety of analytical applications.
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Lab products found in correlation
Nexera x2 uhplc, by Shimadzu (3 mentions)
Tracefinder 3, by Thermo Fisher Scientific (3 mentions)
Ultimate 3000 hplc system, by Thermo Fisher Scientific (2 mentions)
Q exactive, by Thermo Fisher Scientific (2 mentions)
Poroshell 300sb c8 column, by Agilent Technologies (2 mentions)
Ultimate 3000, by Thermo Fisher Scientific (2 mentions)
Jnm ecp500, by JEOL (2 mentions)
Multiskan go, by Thermo Fisher Scientific (2 mentions)
Rf 6000, by Shimadzu (2 mentions)
Kinetex c18, by Phenomenex (1 mentions)
Tracefinder 4, by Thermo Fisher Scientific (1 mentions)
Thermo xcalibur 4, by Thermo Fisher Scientific (1 mentions)
Shodex asahipak nh2p 50 4e, by Resonac (1 mentions)
1260 system, by Agilent Technologies (1 mentions)
Avance 3 system, by Bruker (1 mentions)
C18 column, by Agilent Technologies (1 mentions)
Progenesis qi software, by Waters Corporation (1 mentions)
Inova 500 mhz instrument, by Agilent Technologies (1 mentions)
Zorbax eclipse xdb c18 column, by Agilent Technologies (1 mentions)
Agilent 1290 uhplc system, by Agilent Technologies (1 mentions)
35 protocols using exactive plus orbitrap mass spectrometer
1
Quantitative Metabolite Profiling by HPLC-MS
2
Profiling Plasma Metabolites via LC-MS
3
Metabolic Profiling of Selenium Diets
4
UHPLC-HRMS Analysis of Polar Phenolics
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As previously reported [53 (link)], serum samples were analyzed with UHPLC–HRMS. A binary solvent system consisting of (A) 0.1% formic acid (HCOOH) in acetonitrile (CH3CN) and (B) 0.1% v/v HCOOH was used for chromatographic analysis of polar phenolics, along with an Accucore™ column (C18, 150 × 2.1 mm, 2.6 μm, Thermo Fisher Scientific, Waltham, MA, USA). Gradient elution began with 5% of solvent B for 1.1 min, followed by a rapid increase to 25% in 1.1 min, which then remained isocratic for an additional 1.1 min. After 0.6 min, the concentration of B was raised to 40% and remained at this level for 2.6 min. In the next 3 min, the concentration of B increased to 95%. The initial conditions were then restored in 0.4 min and maintained for 5.6 min for column equilibration, which resulted in a total run time of 18 min; the injection volume was 5 μL. HRMS data were acquired with an Exactive Plus™ Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany), which operated in negative heated electrospray ionization mode (H-ESI). Thermo XCalibur 4.0 was used to handle acquisition, while quantitative results were obtained with TraceFinder™ 4.1 (Thermo Fisher Scientific, San Jose, CA, USA).
5
Quantification of 5-FU Metabolites and PRPP
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Cells were plated in 6‐well plates at 2 × 106 cells per well in standard medium. For 5‐FU metabolites measurement, cells were cultured in medium containing 10 μg/mL 5‐FU for 24 hours. For PRPP measurement, cells were cultured in RPMI 1640 and incubated with [U‐13C6]‐d ‐glucose for 5 minutes. At the end of incubations, cells were rapidly washed two times with cold PBS and extracted with ice‐cold extraction solution composed of 80% Methanol in water (1000 μL/2 × 106 cells). The lysates were vortexed for 10 minutes at 4°C and immediately centrifuged at 15 000 rpm for 15 minutes at 4°C. The supernatants were collected and analyzed by LC‐MS.
For the LC separation, a ZIC‐pHILIC (150 × 2.1 mm, SeQuant, Darmstadt, Germany) with a guard column (20 × 2.1 mm, SeQuant, Darmstadt, Germany) was used. The mobile phase A was 20 mmol/L ammonium carbonate plus 0.1% ammonia hydroxide in water and mobile phase B was acetonitrile. The flow rate was 200 μL/mL and gradient as follows: 0 minutes 80% of B to 25 minutes 20% of B and the column was then re‐equilibrated until 32 minutes at 80% of B. The Exactive Plus Orbitrap mass spectrometer (Thermo Scientific, Carlsbad, CA, USA) was operated in a polarity switching mode.
For the LC separation, a ZIC‐pHILIC (150 × 2.1 mm, SeQuant, Darmstadt, Germany) with a guard column (20 × 2.1 mm, SeQuant, Darmstadt, Germany) was used. The mobile phase A was 20 mmol/L ammonium carbonate plus 0.1% ammonia hydroxide in water and mobile phase B was acetonitrile. The flow rate was 200 μL/mL and gradient as follows: 0 minutes 80% of B to 25 minutes 20% of B and the column was then re‐equilibrated until 32 minutes at 80% of B. The Exactive Plus Orbitrap mass spectrometer (Thermo Scientific, Carlsbad, CA, USA) was operated in a polarity switching mode.
6
Glycosyltransferase Activity Assay
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Standard assays were performed with 1.5 mM 4MU-β-d -Galf acceptor substrate, 40 mM UDP-galactopyranose, purified Glf protein (UDP-galactopyranose mutase from Escherichia coli ; 15.8 μg), 40 mM sodium dithionite (SD), and purified GfsA (4.5 μg), GfsB (4.5 μg), or GfsC (4.5 μg) protein in a total reaction volume of 20 μl. The mixtures were incubated at 30°C for 16 h, and the reaction was stopped by the application of heat (99°C) for 5 min. The supernatants were analyzed by HPLC with an amino column (Shodex Asahipak NH2P-50 4E; Showa Denko, Tokyo, Japan) (250 by 4.6 mm) as previously described (4 (link)). 4-Methylumbelliferyl and p-nitrophenyl derivatives were detected by 300 nm of absorbance. The mass spectra of the enzymatic products of GfsA, GfsB, and GfsC were determined using an Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific).
7
Identification of OsJAZ11 Interactors
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OsJAZ11-GST protein was extracted and immobilized on glutathione-agarose beads as described above. Total protein from 30-day-old OsJAZ11 overexpression lines was isolated using extraction buffer (1X PBS pH 7.4 containing 0.5 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1X plant Protease Inhibitor Cocktail). Extracted protein was incubated with recombinant OsJAZ11-GST protein bound to glutathione-agarose beads at 4 °C overnight. Beads were subsequently washed thrice with 1X PBS and OsJAZ11-bound protein complexes were eluted with 30 mM reduced glutathione (pH 8.0). Eluted proteins were identified by LC–MS using Exactive™ Plus Orbitrap Mass Spectrometer (Thermo Fisher Scientific).
8
HPLC-Orbitrap Analysis of Genistein Metabolites
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HPLC
was carried out using an Agilent 1260 system with a DAD detector,
equipped with a SILGREEN C18 column (4.6 × 250 mm2, 5 μm). The sample volumes of 50 μL were injected and
a flow rate of 1 mL/min was set. The reaction mixtures were separated
using a gradient of HPLC grade water (solvent A) and HPLC grade acetonitrile
(solvent B) as the mobile phase according to the following eluting
program: 0–35 min, linear gradient from 5 to 50% B (v/v); 35–36
min, linear gradient from 50 to 100% B (v/v); 36–40min, held
at 100% B (v/v); 40–42, 100 to 5% B (v/v); and 42–45
min, kept at 5% B (v/v). C18 column was kept at ambient temperature,
and the peaks were detected at a wavelength of 265 nm.
Hydroxylated
products of genistein collected from the C18 column were injected
into a Thermo Exactive Plus Orbitrap mass spectrometer for high-resolution
mass spectrometry (HR-MS) measurement. An ESI source was employed
in a positive ionization mode. Full MS scans were acquired over the
range of m/z 100–1500.
NMR spectra were
recorded on a Bruker 600 MHz AVANCE III system,
as introduced in our previous reports.37 (link)−39 (link) Briefly, the samples
were run in deuterated methanol (CD3OD) at 25 °C. Chemical shifts
were recorded in δ (ppm) with the residual methyl signals in
CD3OD as the references. The NMR assignments of hydroxygenisteins
were based on 1H and 13C chemical shifts.
was carried out using an Agilent 1260 system with a DAD detector,
equipped with a SILGREEN C18 column (4.6 × 250 mm2, 5 μm). The sample volumes of 50 μL were injected and
a flow rate of 1 mL/min was set. The reaction mixtures were separated
using a gradient of HPLC grade water (solvent A) and HPLC grade acetonitrile
(solvent B) as the mobile phase according to the following eluting
program: 0–35 min, linear gradient from 5 to 50% B (v/v); 35–36
min, linear gradient from 50 to 100% B (v/v); 36–40min, held
at 100% B (v/v); 40–42, 100 to 5% B (v/v); and 42–45
min, kept at 5% B (v/v). C18 column was kept at ambient temperature,
and the peaks were detected at a wavelength of 265 nm.
Hydroxylated
products of genistein collected from the C18 column were injected
into a Thermo Exactive Plus Orbitrap mass spectrometer for high-resolution
mass spectrometry (HR-MS) measurement. An ESI source was employed
in a positive ionization mode. Full MS scans were acquired over the
range of m/z 100–1500.
NMR spectra were
recorded on a Bruker 600 MHz AVANCE III system,
as introduced in our previous reports.37 (link)−39 (link) Briefly, the samples
were run in deuterated methanol (CD3OD) at 25 °C. Chemical shifts
were recorded in δ (ppm) with the residual methyl signals in
CD3OD as the references. The NMR assignments of hydroxygenisteins
were based on 1H and 13C chemical shifts.
9
Proteomics of Polarized T Helper Cell Subsets
10
NMR and HRMS Analysis Protocol
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1H NMR and 13C NMR spectra were recorded on a Varian Inova 500 MHz instrument (Varian, Inc) in solutions of (CD3)2SO. HRMS was performed using an Exactive Plus Orbitrap mass spectrometer (Thermo Scientific) with an ESI source.
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