The phytochemical profiling of OIE was analyzed using LC-QTOF-MS/MS in negative mode. The analyses were performed on the Dionex Ultimate 3000 UHPLC system (Dionex, USA) coupled with an electrospray ionization (ESI) tandem mass spectrometer (micrOTOF-Q II) (Bruker, Germany). A sample injection volume of 5 μL was used for chromatographic separation of analytes using a Zorbax SB-C18 (250 mm × 4.6 mm × 3.5 μm (Agilent Technologies, USA)) and a gradient program including deionized water containing 0.1% formic acid (FA) as solvent A and acetonitrile containing 0.1% formic acid (FA) as solvent B. The flow rate of the mobile phase was fixed at 0.8 mL/min and the temperature of the column was fixed at 35°C. The gradient program was optimized by passing through the reservoir 30% B, reaching 80% B at 30 min, and holding until 38 min, reducing at 30% B in 2 min, and holding until the run ended at 45 min. The LC-QTOF data were collected and processed by Compass 1.3 software (Bruker, Germany). Apiin, luteolin, quercetin, apigenin, kaempferol, baicalein, and oroxylin A were used as standard reference compounds. The calibration curves were constructed from peak areas of different concentrations of the reference standard (from 1 μg/mL to 100 μg/mL), and the concentrations of targeted compounds were calculated based on the equation for linear regression obtained from the calibration curves.
Dionex ultimate 3000 uhplc system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, Australia
The Dionex Ultimate 3000 UHPLC system is a high-performance liquid chromatography (HPLC) instrument designed for ultra-high-performance liquid chromatography (UHPLC) applications. The system features a compact, modular design and is capable of operating at pressures up to 1,300 bar (18,900 psi), enabling the use of sub-2 μm particle size columns for improved separation performance and reduced analysis time.
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Lab products found in correlation
Q exactive mass spectrometer, by Thermo Fisher Scientific (7 mentions)
Q exactive, by Thermo Fisher Scientific (7 mentions)
Q exactive hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (6 mentions)
Acquity uplc beh c18 column, by Waters Corporation (5 mentions)
Xcalibur software, by Thermo Fisher Scientific (5 mentions)
Chromeleon 7, by Thermo Fisher Scientific (4 mentions)
Hypersil gold c18 column, by Thermo Fisher Scientific (4 mentions)
Q exactive orbitrap mass spectrometer, by Thermo Fisher Scientific (4 mentions)
Thermo high resolution q exactive focus mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Acclaim 120 c18 column, by Thermo Fisher Scientific (3 mentions)
Q exactive plus hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Q exactive orbitrap, by Thermo Fisher Scientific (3 mentions)
Kinetex c18 column, by Phenomenex (3 mentions)
Ltq orbitrap velos mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Sb aq column, by Agilent Technologies (3 mentions)
Ptfe syringe filters, by Restek (2 mentions)
Sequant zic philic column, by Merck Group (2 mentions)
Sequant zic philic guard column, by Merck Group (2 mentions)
Zic philic column, by Merck Group (2 mentions)
Hss t3 column, by Waters Corporation (2 mentions)
171 protocols using dionex ultimate 3000 uhplc system
1
Phytochemical Profiling of Olea indica Extract
2
Coomassie Protein Digestion and MS/MS Analysis
3
UHPLC-based Glucose Quantification
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Quantification of glucose resulting from reactions with the Celluclast® enzyme cocktail was achieved using a Dionex Ultimate 3000 UHPLC system (Dionex, Sunnyvale, CA, USA) equipped with a Rezex ROA-Organic Acid H + (8%), 300 × 7.8 mm analytical column and a SecureGuard Carbo-H + 4 × 3.0 mm guard column (Phenomenex, Torrance, CA, USA) operated at 65 °C. Sample components were eluted isocratically over 22 min using 5 mM sulfuric acid as mobile phase with a flow rate of 0.6 mL/min. Products were detected using a refractive index (RI) detector 101 (Shodex, Tokyo, Japan) and data collection and analysis were carried out with the Chromeleon 7.0 software.
4
Comparative Proteomic Analysis of P. gingivalis Strains
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Aliquots (2 mL) of growth medium collected from cultures of P. gingivalis strains ATCC 33277, ∆ppad (ATCC 33277), W83 and Δppad (W83) were lyophilized in an Alpha 1-2 lyophilizer (Martin Christ, Osterode am Harz, Germany) and then dissolved in 100 µL of ultra-pure water to achieve a 20-fold concentration. Three biological replicates were prepared. Samples for electrophoresis were prepared by mixing 15 μL of concentrated supernatant with 15 μL of loading buffer for subsequent SDS-PAGE electrophoresis followed by incubation at 95 °C for 5 min. After electrophoretic separation using the Laemmli system [80 (link)], protein detection was carried out with Coomassie Brilliant Blue G-250 staining and specific protein bands were excised from the gel. These gel samples were subjected to tryptic digestion followed by protein identification with LC-MS/MS using the Dionex UltiMate 3000 UHPLC system (Dionex, Carlsbad, CA) and the HCTUltra ETDII ion-trap mass spectrometer equipped with an electrospray ionization ion source (Bruker, Bremen, Germany), as described previously [81 (link)]. Protein identification was then performed using a SwissProt protein database search (560,118 sequences for all entries, including 336,487 sequences for bacterial proteins) with an in-house Mascot server (v.2.3.0, Matrix Science, London, UK).
5
Targeted Metabolomics Analysis by UHPLC-Orbitrap
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Analysis was performed on a Dionex Ultimate 3000 UHPLC system (Dionex, now Thermo Fisher Scientific, Hemel Hempstead, UK) interfaced via an electrospray ionisation (ESI) probe to an Orbitrap Elite MS (Thermo Fisher Scientific). Chromatographic separation was carried out on a Hypersil Gold reversed phase C18 column (1.9 µm particle size, 50 x 2.1 mm, Thermo Fisher Scientific, UK). Details of the mobile phase and gradients employed are given in Supplemental Materials and Methods. MS analysis on the Orbitrap Elite was performed in the positive-ion mode with five scan events, one high resolution (120,000 full width at half maximum height at m/z 400) scan over the m/z range 400 – 610 in the Orbitrap and four MS3 scans performed in parallel in the linear ion trap (LIT). Mass accuracy in the Orbitrap was typically < 5 ppm. More details of the scan events are provided in Supplemental Materials and Methods. Injection volumes were 35 µL for plasma extracts and at 90 µL for amniotic fluid and placental extracts.
6
UHPLC-Q Exactive Focus LC-MS Protocol
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The Thermo Scientific Dionex Ultimate 3000 UHPLC system hyphenated with a Thermo Q exactive focus machine used was already reported. For the analysis, 2 mg of each extract were first dissolved in 2 mL of ethanol, then filtered (PTFE filter) and finally 10 μL were injected in the instrument, with all specifications set as previously reported (Salgado et al., 2017 (link); Torres-Benitez et al., 2017 (link)).
7
Quantification of Plasma TMAO Levels
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The plasma TMAO concentrations were quantified using liquid chromatography-mass spectrometry that employed a Q Exactive instrument (Thermo Scientific, San Jose, CA) equipped with a Dionex Ultimate 3000 UHPLC system (Thermo Scientific), and separation was carried out at 30 °C in an Xbridge amide column (4.6 × 150 mm, 3.5 mm, Waters, Milford, MA). The mobile phase consisted of a combination of 5% acetonitrile containing 5 mM ammonium acetate in MilliQ water (solution A) and 5 mM ammonium acetate in acetonitrile (solution B). The chromatogram was run under isocratic conditions at a flow rate of 0.7 mL/min, as follows: A/B = 95/5. A heated electrospray ionization (HESI) ion source was used for the ionization. The HESI parameters were optimized as follows: sheath gas flow rate 53 units, auxiliary gas unit flow rate 14, capillary temperature 269 °C, auxiliary gas heater temperature 45 °C, spray voltage 2500 V, and S lens RF level 55.
8
UHPLC-QTOF High-Resolution Metabolomics
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Standardized microbial extracts were dissolved in 1 mL of methanol (LCMS grade, CarloErba, Val de Reuil, France) and filtered with 0.22 μm PTFE syringe filters (Restek, Bellefonte, PA, USA). A Dionex Ultimate 3000 UHPLC system (Thermo Scientific, Waltham, MA, USA) coupled to a Bruker Impact II QtoF mass spectrometer (Bruker, Billerica, MA, USA) was used for high-resolution analysis. The separation was performed on a Phenomenex Kinetex phenyl hexyl analytical column (1.7 μm, 150 × 2.1 mm, 1.7 µm) (Phenomenex, Torrance, CA, USA) with two solvents, ACN (MS grade, CarloErba, Val de Reuil, France) (phase A), and milli-Q water (phase B), each containing 0.1% formic acid (FA) (analytical grade 99% purity, CarloErba, Val de Reuil, France). Elution was performed with a linear gradient from 0 to 100% B for 8 min with a flow rate of 0.5 mL/min. The main MS data acquisition parameters were as follows: an acquisition in positive mode (ESI+; 20–40 eV) and in the range of 20 to 1200 Da for MS1 spectra, a collision energy of 40 eV, an acquisition speed of 4 Hz, and the selection of the 5 major MS1 precursors for the recording of MS2 spectra.
9
Quantifying Monoclonal Antibody Oxidation
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The separation of oxidized species of LMU1 was performed on a Thermo Scientific™ Dionex™ UltiMate™ 3000 UHPLC system equipped with a VWD-3400RS UV/Vis absorbance detector using a MabPac HIC-20 column (4.6 × 250 mm), all from Thermo Fisher Scientific (Waltham, MA, USA). According to Baek et al. [29 ] the mobile phase A contained 2 M ammonium sulfate and 100 mM sodium phosphate, pH 7.0, whereas mobile phase B solely consisted of 100 mM sodium phosphate, pH 7.0. Prior to analysis, the samples were diluted to a mAb concentration of 5 g/L with mobile phase A, and 5 μL were injected. Starting with 60% B at a flow rate of 0.5 mL/min for 2 min, a linear gradient from 60% to 100% B in 28 min was then performed to separate the oxidation variants of LMU1. The elution of the samples was detected by absorption at 280 nm. The chromatograms were integrated using Chromeleon™ 7.2.7 (Thermo Fisher Scientific, Waltham, MA, USA). Because of the different extinction coefficients of the oxidized species, we used Equation (1) for the determination of the amount of fully oxidized mAb, adapted from Reference [30 ]:
RFI/O UV 280 nm: 1.49. For the calibration data seeFigure S1 in the Supplementary Materials .
RFI/O UV 280 nm: 1.49. For the calibration data see
10
Quantitative LC-MS/MS for Metabolite Analysis
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Briefly, the LC-MS/MS experiments were performed on the Dionex UltiMate 3000 Uhplc system coupled with Q Exactive mass spectrometer (Thermo Fisher Scientific, CA, USA) operating in data-dependent acquisition (DDA) mode. Samples were injected onto a Hypersil GOLD HPLC column (50×2.1 mm, 1.9 μm). The mobile phase consisted of a gradient system of (A) 10 mM ammonium formate in water and (B) 10 mM ammonium formate in methanol: 0–2 min, 5% B; 2–5 min, 5–30% B; 5–19 min, 30–99% B; 19–22 min, 99% B; and 22.1–25 min, 5% B.
Compound ionization was conducted as the following parameters: Q-Exactive mass spectrometer was operated in positive/negative polarity mode with spray voltage 3.5 kV/3.2 kV, capillary temperature of 320°C, sheath gas flow rate 30 psi and aux gas flow rate 10 arb. Samples were analyzed using liquid chromatography-high resolution mass spectrometry (LC-HRMS) in full scan + data dependent MS2 mode with a scan range from 100–1000 m/z at a resolution of 70,000, followed by data dependent MS/MS (dd-MS/MS) with a normalized collision energy of 30 and at a resolution of 17,500. To avoid instrument drift, fourteen quality control (QC) samples were preprocessed as the samples for data quality assessment.
Compound ionization was conducted as the following parameters: Q-Exactive mass spectrometer was operated in positive/negative polarity mode with spray voltage 3.5 kV/3.2 kV, capillary temperature of 320°C, sheath gas flow rate 30 psi and aux gas flow rate 10 arb. Samples were analyzed using liquid chromatography-high resolution mass spectrometry (LC-HRMS) in full scan + data dependent MS2 mode with a scan range from 100–1000 m/z at a resolution of 70,000, followed by data dependent MS/MS (dd-MS/MS) with a normalized collision energy of 30 and at a resolution of 17,500. To avoid instrument drift, fourteen quality control (QC) samples were preprocessed as the samples for data quality assessment.
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