UHPLC-MS/MS was used for the analysis of serum metabolites. The chromatographic column used was the Acquity™ UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 µM) (Waters, United States). Both the positive and negative modes were selected as analytes. The UHPLC-MS/MS analysis uses the Q Exactive Plus High Resolution Mass Spectrometer (THERMO, United States) and was equipped with Ultimate 3,000 (DIONEX, TERMO, United States). The mass spectrometer operates in full mass spectrometry mode in the positive ionization mass range of 100–1500 M/Z. The chromatographic condition and the serum sample gradient were followed as previously described (Ren et al., 2018 (link)). The optimized chromatographic conditions were as follows: at the flow rate of 0.3 ml/min with a mobile phase consisting of isopropanol/acetonitrile 50:50 (V/V) with 0.1% formic acid (mobile phase A) and 0.1% formic acid solution (mobile phase B). Gradient elution process: 0–2 min, 2% A; 2–22 min, 2–99% A; 22–25 min, 99% A; 25.1–30 min, 2% A. The injection volume was 10 µl. Between two injections, the sampling needle was washed with 300 µl washing solution (methanol/water 50/50 V/V) once. The mass spectrometer was operated in full MS mode in the range of 100–1500 m/z positive ionization mass.
Q exactive plus high resolution mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Q Exactive Plus High Resolution Mass Spectrometer is a high-performance mass spectrometer designed for accurate mass measurement and high-resolution analysis. It provides precise quantification and identification of small molecules, peptides, and proteins.
Automatically generated - may contain errors
Lab products found in correlation
Acquity uplc beh c18 column, by Waters Corporation (3 mentions)
Ultimate 3000, by Thermo Fisher Scientific (2 mentions)
Dionex ultimate 3000 rslc, by Thermo Fisher Scientific (1 mentions)
Fluka silica gel 60 plates, by Merck Group (1 mentions)
Dionex ultimate 3000 rslc nano uplc system, by Thermo Fisher Scientific (1 mentions)
6 protocols using q exactive plus high resolution mass spectrometer
1
UHPLC-MS/MS Analysis of Serum Metabolites
2
Quantification of PMO-Gly in Mouse Tissues
3
UHPLC-MS/MS Analysis of Complex Metabolites
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UHPLC-MS/MS analysis was performed using Q Exactive Plus High Resolution Mass Spectrometer (THERMO, USA) equipped with Ultimate 3000 (DIONEX, THERMO, USA). Acquity™ UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) (Waters, USA) was used. The optimized chromatographic conditions were achieved at a flow rate of 0.3 mL/min with a mobile phase consisting of isopropanol/facetonitrile 50:50 (V/V) with 0.1% formic acid (mobile phase A) and 0.1% formic acid solution (mobile phase B). The gradient elution process was: 0–2 min, 2% A; 2–22 min, 2–99% A; 22–25 min, 99% A; 25.1–30 min, 2% A. The injection volume was 10 μL. The sampling needle was washed with 300 μL of wash (methanol/water 50/50 V/V) once between two injections. The mass spectrometer was operating in full MS mode at a mass range of 100–1500 m/z in positive ionization.
4
UHPLC-MS/MS Analysis of Metabolites
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UHPLC-MS/MS analysis was performed using Q Exactive Plus High Resolution Mass Spectrometer (THERMO, USA) equipped with Ultimate 3000 (DIONEX, THERMO, USA). Acquity™ UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) (Waters, USA) was used. The chromatographic condition in reference was used [35 (link)]. The mobile phase consisting of isopropanol/facetonitrile 50:50 (V/V) with 0.1% formic acid (mobile phase A) and 0.1% formic acid solution (mobile phase B). The gradient elution process was 0–2 min, 2% A; 2–22 min, 2–99% A; 22–25 min, 99% A; 25.1–30 min, 2% A. The injection volume was 10 μL. The mass spectrometer was operating in full MS mode at a mass range of 100–1500 m/z in positive ionization.
5
NMR and Mass Spectrometry Analysis
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All reagents and chemicals were procured from commercial suppliers, namely Sigma-Aldrich S.A. and Riedel-de Haën, Sofia, Bulgaria, and used without further purification. NMR spectral data were acquired using a Bruker Avance Neo 400 spectrometer (BAS-IOCCP–Sofia, Bruker, Billerica, MA, USA). The 1H-NMR and 13C-NMR spectra for all compounds were obtained in DMSO-d6 at 400 MHz and 101 MHz, respectively. Chemical shifts are reported in relative ppm and were calibrated with respect to tetramethylsilane (TMS) at δ = 0.00 ppm, serving as the internal standard, with coupling constants denoted in Hz. NMR spectra were recorded at room temperature (approximately 295 K). For mass spectrometry (MS) analysis, a Q Exactive Plus high-resolution mass spectrometer (HRMS) with a heated electrospray ionization source (HESI-II) from Thermo Fisher Scientific, Inc., Bremen, Germany, was employed. The MS system was equipped with a Dionex Ultimate 3000RSLC ultrahigh-performance liquid chromatography (UHPLC) system from Thermo Fisher Scientific, Inc., Waltham, MA, USA. Thin-layer chromatography (TLC) was conducted using 0.2 mm Fluka silica gel 60 plates obtained from Merck KGaA, Darmstadt, Germany.
6
Peptide Separation and Characterization by UPLC-MS
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The peptide mixture was subjected to reverse phase chromatography on a Dionex Ultimate 3,000 RSLC nano-UPLC system (Thermo Fisher Scientific, Inc.) in-line coupled to a Q-Exactive Plus high-resolution mass spectrometer (Thermo Fischer Scientific, Inc.). Peptides (2 µg) resuspended were first trapped on a precolumn (C18 PepMap 100, 5 µm, 100 A, 300 µm inner diameter × 5 mm; Thermo Fisher Scientific, Inc), then separated using an EASY-Spray PepMap RSLC C18 capillary column (2 µm, 15 cm × 50 µm; Thermo Fischer Scientific, Inc.) with a 250-min elution gradient at 250 nl/min. The mobile phases were: A) 2% acetonitrile and 0.1% formic acid in water; and B) 90:10 (v:v) acetonitrile:water and 0.1% formic acid in water. The mass spectrometer was operated in positive data-dependent acquisition mode and the full MS range was 300-1,800 m/z. A total of 10 of the most intense ions were isolated in the quadrupole and fragmented under higher-energy collisional dissociation with a normalized collision energy of 27%. Precursor ions were measured at a resolution of 70,000 (at 200 m/z) and the fragments were measured at 17,500. Only ions with charge states ≥2 were fragmented with an isolation window of 2 m/z.
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