Hydroxylation assays were performed in triplicate under normoxic conditions using liquid chromatography electrospray MS (LC-ESI-MS) (Figure 3 , Figure 4 ). Conditions: 1 μm TgPhyA or DdPhyA, 100 μm full-length DdSkp1 or full-length TgSkp1 substrate, 50 μm (NH4)2Fe(II)(SO4)2 (Sigma-Aldrich), 1 mm sodium l -ascorbate (Sigma-Aldrich), and 500 μm 2-oxoglutarate disodium salt (Sigma-Aldrich) in HEPES (100 mm ), pH 7.6. Mixtures were incubated at 37 °C for 1 h, or at specific time points for time course assays, and quenched using an equal volume of 1% (v/v) aqueous formic acid (Sigma-Aldrich). Reaction mixtures were analyzed using a Xevo G2-S Q-TOF mass spectrometer equipped with an electrospray source (Waters®) coupled to a Waters® ACQUITY UPLC System, unless otherwise specified. Instrument control and data processing were performed using MassLynx V4.1 software. An AerisTM 3.6-μm WIDEPORE C4 200 Å (Phenomenex) 4.6 × 50 mm column was used for separation.
G2 s q tof mass spectrometer
Manufactured by Waters Corporation
Sourced in United States
The G2-S Q-TOF mass spectrometer is a high-resolution, accurate-mass instrument designed for advanced analytical applications. It utilizes quadrupole and time-of-flight technology to provide precise mass measurements and detailed structural information about molecular compounds. The core function of the G2-S Q-TOF is to enable accurate mass determination and fragmentation analysis of complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Aqueous formic acid, by Merck Group (1 mentions)
Sodium l ascorbate, by Merck Group (1 mentions)
Nh4 2fe 2 so4 2, by Merck Group (1 mentions)
Acquity uplc system, by Waters Corporation (1 mentions)
Avance 2 400 spectrometer, by Bruker (1 mentions)
Masslynx 4, by Waters Corporation (1 mentions)
Acquity, by Waters Corporation (1 mentions)
Beh glycan column, by Waters Corporation (1 mentions)
Masslynx software, by Waters Corporation (1 mentions)
Beh c18 column, by Waters Corporation (1 mentions)
Uv 1800, by Shimadzu (1 mentions)
5 protocols using g2 s q tof mass spectrometer
1
Hydroxylation Assays with LC-ESI-MS
2
NMR and Mass Spectrometry Analysis of Organic Compounds
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1H (400 MHz), 31P (162 MHz) and 13C (101 MHz) NMR spectra were recorded on a Bruker Avance II 400 spectrometer at 298 K. Chemical shifts are reported in parts per million (ppm) and referenced to deuterated solvent residual peaks (CDCl3: 1H δ 7.26, 13C{1H} δ 77.16 ppm) and coupling constants (J) are reported in Hertz (Hz). High resolution electrospray ionization mass spectra (HR ESI-MS) were obtained on a Xevo G2-S QTOF mass spectrometer coupled to the Acquity UPLC Class Binary Solvent manager and BTN sample manager (Waters, Corporation, Milford, MA). Elemental analyses were determined on a Thermo Scientific Flash 2000 Organic Elemental Analyzer.
3
N-Glycan Analysis by UPLC-QTOF MS
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The prepared N-glycans were analyzed by a Waters Acquity ultra-performance liquid chromatography instrument equipped with a fluorescence detector and a Xevo-G2S qTOF mass spectrometer. The system was controlled by MassLynx 4.2 (Waters, Milford, MA, USA). Separations were performed by a Waters BEH Glycan column, 100 × 2.1 mm i.d., 1.7 μm particles, using a linear gradient of 75–55% acetonitrile (Buffer B) at 0.4 mL/min in 42 min, using 50 mM ammonium formate pH 4.4 as Buffer A. An amount of 5 μL of sample was injected using partial loop mode in all runs. The sample manager temperature was 15 °C, and the column temperature was 60 °C during each analysis. The fluorescence detection excitation and emission wavelengths were λex = 308 nm and λem = 359 nm. During the MS analysis, 2.2 kV electrospray voltage applied to the capillary. The desolvation temperature was set to 120 °C, while the desolvation gas flow was 800 L/hr. Mass spectra were acquired using positive ionization mode over the range of 500–2000 m/z. MS/MS fragments were obtained using 45 kV collision energy during the analysis.
4
UPLC-QTOF-MS Analysis of Compounds
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LC-MS analysis was performed on an ultraperformance liquid chromatography (UPLC) system coupled to a Xevo G2-S QTof mass spectrometer equipped with an electrospray ionization (ESI) source (Waters, Milford, MA, USA). Separation was performed on a BEH C18 column (Waters, Wexford, Ireland; 50 × 2.1 mm i.d., 1.7 µm particle size) at 40°C with a gradient elution programmed at constant flow rate (0.3 ml.min−1). The extract was analyzed at concentration of 1 mg.ml−1 injected (5 µl) simultaneously with leucine-enkephalin (reference compound). Mobile phase consisted of 0.1% formic acid aqueous solution (A) and 0.1% formic acid in acetonitrile (B). A linear gradient was performed ranging from 5 to 95% B in 10 min. ESI source was operated in positive mode over a wide mass range (m/z 50–1,200) with a scan time of 0.1 s. The source temperature was set at 150°C with a cone gas flow of 20 L h−1. Desolvation gas flow was set at 600 L.h−1 at a temperature of 450°C. Capillary was set at 3.0 kV with cone voltage at 20 V. MassLynx Software (Waters, Milford, MA, USA) was used for data acquisition and processing.
5
Characterization of Organic Compounds
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Melting points were
determined in open glass capillaries and were reported uncorrected. 1H NMR was recorded on a Jeol ECS, 400 MHz, using CDCl3 as the solvent. Tetramethylsilane was taken as an internal
standard, and the chemical shifts were recorded in δ ppm. Resonance
multiplicities were described as s (singlet), d (doublet), t (triplet),
q (quartet), and m (multiplet). IR spectra were recorded on a PerkinElmer
Spectrum Two spectrometer using KBr pellet. Mass spectra were recorded
on a Xevo G2-S Q-Tof mass spectrometer (Waters), capable of recording
high-resolution mass spectrum (HRMS) in the electrospray ionization
(ESI) mode. An Elma S 70 H ultrasonic unit with 37 kHz output frequency
was employed for ultrasonication. UV–visible spectra and fluorescence
spectra were recorded on Shimadzu UV-1800 in standard 3.5 mL quartz
cells with 10 mm path length and FluoroMax 4 C.L. system, respectively.
determined in open glass capillaries and were reported uncorrected. 1H NMR was recorded on a Jeol ECS, 400 MHz, using CDCl3 as the solvent. Tetramethylsilane was taken as an internal
standard, and the chemical shifts were recorded in δ ppm. Resonance
multiplicities were described as s (singlet), d (doublet), t (triplet),
q (quartet), and m (multiplet). IR spectra were recorded on a PerkinElmer
Spectrum Two spectrometer using KBr pellet. Mass spectra were recorded
on a Xevo G2-S Q-Tof mass spectrometer (Waters), capable of recording
high-resolution mass spectrum (HRMS) in the electrospray ionization
(ESI) mode. An Elma S 70 H ultrasonic unit with 37 kHz output frequency
was employed for ultrasonication. UV–visible spectra and fluorescence
spectra were recorded on Shimadzu UV-1800 in standard 3.5 mL quartz
cells with 10 mm path length and FluoroMax 4 C.L. system, respectively.
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