Profile of metabolites were tested by Dionex Ultimate 3000 RS UHPLC system (Thermo Fisher Scientific) with heated electrospray ionization in positive and negative modules. Potential metabolites was obtained and identified by progenesis QI software (Waters Corporation), based on Human Metabolome Database (HMDB1 ). QC samples were injected every 10 samples for accessible repeatability. Details of the parameters in sample detection and data process can be referred to Supplementary Material .
Dionex ultimate 3000 rs uhplc system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Dionex Ultimate 3000 RS UHPLC system is a high-performance liquid chromatography (HPLC) instrument designed for efficient and precise separation and analysis of a wide range of analytes. It features a modular design, advanced control software, and high-resolution detection capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Progenesis qi software, by Waters Corporation (2 mentions)
Q exactive quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Acquity uplc hss t3 column, by Waters Corporation (1 mentions)
Progenesis qi, by Waters Corporation (1 mentions)
Dataanalysis 4, by Bruker (1 mentions)
Vanguard pre column, by Waters Corporation (1 mentions)
Acquity uhplc beh amide column, by Waters Corporation (1 mentions)
Amazon speed, by Bruker (1 mentions)
Q exactive plus orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
S500 spectrometer, by Agilent Technologies (1 mentions)
9 protocols using dionex ultimate 3000 rs uhplc system
1
Metabolite Profile Analysis by UHPLC-HRMS
2
Profiling Rice Shoot Metabolites under Salt Stress
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Metabolites were extracted from each rice shoot sample (approximately 80 mg) using methanol. At Shanghai Lu-Ming Biotech Co., Ltd. (Shanghai, China), metabolites were measured with a Dionex Ultimate 3000 RS UHPLC system coupled to a Q-Exactive quadrupole-Orbitrap mass spectrometer (MS) (Thermo Fisher Scientific, Bremen, Germany). An ACQUITY UPLC HSS T3 column (Waters, Milford, MA, USA) was employed in both positive and negative modes.
The LC-MS raw data were analyzed and normalized by progenesis QI software (v2.3) (Nonlinear Dynamics, Newcastle, UK). The Human Metabolome Database (HMDB) (https://hmdb.ca/ ), Lipidmaps (v2.3) (https://lipidmaps.org/ ), and METLIN Database (https://ngdc.cncb.ac.cn/databasecommons/database/id/5907 ) were all used to identify metabolites. Metabolites with a score >36 were reliable, and positive and negative data were combined. A principal component analysis (PCA) of metabolites from all rice seedling samples and QC samples was conducted. Metabolites with variable importance in projection (VIP) of the Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) mode > 1 and P < 0.05 were considered to be differentially abundant metabolites (DAMs). Fold change (the metabolite quantity under salt stress/normal condition) > 1 indicated up-regulation, whereas FC < 1 indicated down-regulation. KEGG pathway enrichment analysis of DAMs was also conducted.
The LC-MS raw data were analyzed and normalized by progenesis QI software (v2.3) (Nonlinear Dynamics, Newcastle, UK). The Human Metabolome Database (HMDB) (
3
Metabolic Profiling by LC-MS
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LC-MS was performed as previously described in detail (21 (link), 22 (link)). A DionexUltimate 3000 RS UHPLC system coupled with mass spectrometer equipped with electrospray ionization (ESI) source (Thermo Fisher Scientific, Waltham, MA, USA) was utilized to detect the metabolic profiling. The ACQUITY UPLC HSS T3 column (100 × 2.1 mm, 1.8 μm) was applied in both positive and negative modes. The gradient elution system was consisted of (A) (water with 0.1% formic acid, v/v) and (B) (acetonitrile containing 0.1% formic acid, v/v). The gradient elution was 5% B in 0.01min, 5% B in 2min, 30% B in 4min, 50% B in 8min, 80% B in 10min, 100% B in 14min, 100% B in 15min, 5% B in 15.1min and 5% B in 14min. Progenesis QI was applied to analyze LC-MS raw data (Waters Corporation, Milford, USA).
4
Chitosan Oligomers Separation by UHPLC-ELSD-ESI-MS
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Chitosan oligomers were separated using a Dionex Ultimate 3000RS UHPLC system (Thermo Fisher Scientific, Waltham, USA) coupled to an evaporative light scattering detector (Model Sedex 90LT, Sedere, Alfortville Cedex, France) and an ESI-MSn-detector (amaZon speed, Bruker, Bremen, Germany). Separation of the oligomers was achieved by hydrophilic interaction liquid chromatography (HILIC) using an Acquity UHPLC BEH Amide column (1.7 μm, 2.1 mm × 150 mm; Waters Corporation, Milford, USA) in combination with a VanGuard pre-column (1.7 μm, 2.1 mm × 5 mm; Waters Corporation, Milford, USA). The samples were split between ELSD and ESI-MSn detectors using a 1:1 splitter (Accurate, Dionex Corporation, Sunnyvale, USA). All of the used UHPLC-ELSD-ESI-MSn methods were based on the ones described by Cord-Landwehr et al. [17 (link)]. The injection volume was always 1 μl for undiluted samples and 2 μl for samples that were diluted with equal parts of 0.5 M NaOH to stop an enzymatic reaction. Data Analysis 4.1 software (Bruker, Bremen, Germany) was used for analysis of the results.
5
Purification and Characterization of Organic Compounds
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The chemicals were purchased from Sigma-Aldrich Co., LLC (Prague, Czech Republic) and were used without additional purification. Analytical thin-layer chromatography was carried out using plates coated with silica gel 60 with the fluorescent indicator F254 (Merck, Prague, Czech Republic). The thin-layer chromatography (TLC) plates were visualized by exposure to ultraviolet light (254 nm) or by the detection reagent ninhydrin. Column chromatography was performed using silica gel 100 at atmospheric pressure (70–230-mesh ASTM, Fluka, Prague, Czech Republic). The NMR spectra were all recorded on a Varian S500 spectrometer (500 MHz for 1H and 126 MHz for 13C). Chemical shifts are reported in δ ppm referenced to residual solvent signals (for 1H NMR and 13C NMR: chloroform-d (CDCl3; 7.26 (D) or 77.16 (C) ppm). A CEM Explorer SP 12 S was used for the MW-assisted reaction. The final compounds were analyzed by LC-MS with a Dionex Ultimate 3000 RS UHPLC system coupled with a Q Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) to obtain high-resolution mass spectra. Gradient LC analysis with UV detection (254 nm) confirmed >95% purity.
6
Quantitative UHPLC Analysis of Phytochemicals
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Quantitative analysis was conducted on a separate Dionex UltiMate 3000 RS UHPLC system (Thermo Fisher Scientific), comprising the same modules as for qualitative analysis without coupling to MS. All chromatographic parameters were identical, with extracted quantification wavelengths from DAD-UV detection being 318 nm for stilbenes and 424 nm for anthranoids. Freeze-dried extracts were redissolved in concentrations of 2 mg/mL, with water as solvent for dry extracts from infusion and decoction, and 50% (v/v) ethanol for macerates. Reference substances were used in 1 mg/mL methanolic stock solutions and subsequent serial dilutions (1:10, 1:100, 1:1000). Injection volumes were 1 and 5 µL for samples and 1 to 5 µL for references according to desired concentration. All measurements were performed in triplicate and quantified substances calculated for their AUC arithmetic means.
7
Untargeted Metabolomics Analysis Pipeline
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The LC–MS experiment and analyses of untargeted metabolomics were performed as described previously [22 (link)]. Briefly, the cortex sample underwent a series of complex processing steps, and then a Dionex Ultimate 3000 RS UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze the metabolic profile. The acquired LC–MS raw data were analyzed by progenesis QI software (Waters Corporation Milford, Milford, MA, USA). PCA and (O) PLS DA were carried out to visualize the metabolic alterations among experimental groups. The differential metabolites were selected on the basis of the combination of a statistically significant threshold of variable influence on projection (values obtained from the OPLS-DA model and p values from a two-tailed Student’s t test on the normalized peak areas, where metabolites with VIP values larger than 1.0 and p values less than 0.05 were considered differential metabolites). Heatmaps and hierarchical cluster analyses were conducted using MeV version 4.6.0. Cytoscape software package version 3.2.0 (National Institute of General Medical Sciences, Bethesda, MD, USA) and the KEGG database was used to plot the correlation networks and identify metabolic pathways.
8
Metabolic Profiling via LC-MS
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100 μl of the samples were added to the internal standard (L-2-chlorophenylalanine, 0.3 mg/ml, methanol) and vortexed for 10 s. And then 300 μl of protein precipitant was added to methanol-acetonitrile and vortexed for 1 min. The extract was sonicated in an ice-water bath for 10 min, and then left for 2 h at −20°C. The supernatant was separated by centrifugation for 10 min and stored at −80°C until LC-MS analysis.
A Dionex Ultimate 3000 RS UHPLC system fitted with Q-Exactive quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, United States) was used to analyze the metabolic profiling. An ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.8 μm) 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). The flow rate was 0.4 ml/min and the column temperature was 45°C. The mass range was from m/z 66.7 to 1,000.5 during the analysis. The resolution was set at 70,000 for the full MS scans and 35,000 for HCD MS/MS scans. The collision energy was set at 10, 20 and 40 eV. The QCs were injected at regular intervals (every 10 samples) throughout the analytical run to provide a set of data from which repeatability can be assessed.
A Dionex Ultimate 3000 RS UHPLC system fitted with Q-Exactive quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, United States) was used to analyze the metabolic profiling. An ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.8 μm) 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). The flow rate was 0.4 ml/min and the column temperature was 45°C. The mass range was from m/z 66.7 to 1,000.5 during the analysis. The resolution was set at 70,000 for the full MS scans and 35,000 for HCD MS/MS scans. The collision energy was set at 10, 20 and 40 eV. The QCs were injected at regular intervals (every 10 samples) throughout the analytical run to provide a set of data from which repeatability can be assessed.
9
Fecal Metabolomics Profiling in Mice
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The non-targeted metabolomics technique was used to analyze the endogenous metabolites of mice feces. In brief, a 60 mg fecal sample of each mouse was prepared, and an ice-cold mixture of methanol and water (4:1 v/v) was added. After freezing at −20 °C for 5 min, the samples were ground at 60 HZ for 2 min, and then quickly transferred to the ice bath for ultrasonic treatment for 10 min. Subsequently, fecal samples were stored at −20°C for 30 min. The samples were centrifuged at 13,000 rpm at 4 °C for 15 min, and then a 400 μL mixture of methanol and water (1:4, v/v) was added to each sample, followed by being vortexed for 30 s and then placed at −20°C for 2 h. The samples were centrifuged at 13,000 rpm at 4 °C for 10 min, and 150 μL of each sample was collected and filtered through a 0.22 μm microfilter to remove impurities. A Dionex Ultimate 3000 RS UHPLC system fitted with a Q Exactive quadrupole Orbitrap mass spectrometer equipped with a heated electrospray ionization (ESI) source (Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze the metabolic profile.
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