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Ultimate 3000 ultra high performance liquid chromatograph

Manufactured by Thermo Fisher Scientific
Sourced in United States

The UltiMate 3000 Ultra High-performance Liquid Chromatograph is a highly sensitive and precise analytical instrument designed to separate, identify, and quantify a wide range of chemical compounds. It utilizes advanced liquid chromatography technology to achieve efficient and reproducible separations, enabling researchers and analysts to obtain accurate and reliable data.

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3 protocols using ultimate 3000 ultra high performance liquid chromatograph

1

Ultrapure LC-MS/MS Metabolomics Analysis

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The UltiMate 3000 Ultra High-performance Liquid Chromatograph (Thermo Fisher, USA) and Thermo Q-Exactive Orbitrap Mass Spectrometer were used for ultrapure LC-MS/MS analysis (Thermo Fisher). Chromatographic separations were performed on Waters ACQUITY UPLC HSS T3 (100 mm × 2.1 mm × 1.8 μm). Thereafter, all the raw data were fed into Progenesis QI (Waters, Milford, MA, USA) and SIMCA-P14.0 (Umetrics AB, Umea, Vasterbotten, Sweden), software for further analysis [15 (link)]. The partial least-squares discriminant analysis (PLS-DA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) supervised pattern recognition methods were applied to identify the overall metabolic differences among the three groups and the variable importance in the projection (VIP) was used to identify characteristic metabolites in the three groups. Distinct metabolites were analyzed based on VIP > 1.0 and P < 0.05. Further information is provided in the Supplemental Materials and Methods.
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2

Quantitative LC-MS/MS Analysis of Compounds

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LC-MS/MS analysis was conducted on an Ultimate 3000 Ultra High-Performance Liquid Chromatograph (UHPLC) coupled to a Thermo TSQ Altis Tandem Quadrupole Mass Spectrometer (Thermo Scientific, San Jose, CA). Chromatographic separation was achieved with an ACQUITY Amide BEH column (1.7 μm, 2.1 × 150 mm) maintained at 30 °C (Waters, Milford, MO). Mobile phase compositions for solvents A and B consisted of ACN/H2O (95:5, v/v) and (50:50, v/v) respectively, with 10 mM ammonium acetate. The gradient profile had a flow rate of 0.4 mL min−1 and ramped from 0.1 to 30% B in 5 min, from 30 to 90% B in 0.1 min, held at 90% B for 1 min, dropped from 90 to 0.1% B in 0.4 min, and held 0.1% B for 1.5 min. Total chromatographic run time was 8.0 min. The injection volume was 2 μL. All analytes were detected using positive ionization mode and SRM. Electrospray ionization (ESI) source conditions are included in supporting information (SI) Table S1. Collision energies and RF lens voltage were optimized for each reference standard and shown in SI Table S2 along with the precursor to product ion transitions. Data collection and analysis was done using Xcalibur 4.2 Qual and Quan Browser (Thermo Scientific, San Jose, CA).
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3

Metabolomic Profiling of Serum and Cells

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Serum and cell metabolites were detected by coupling Ultimate 3000 Ultra-High Performance Liquid Chromatograph (UHPLC) and Q Exative Quadrupole-Electrostatic Field Orbitrap Mass Spectrometer from Thermo Fisher. The raw data were normalized for principal component analysis (PCA). Orthogonal Partial Least Squares-Discriminant Analysis was selected to characterize the differences between groups. Intracellular NAD+/NADH levels were detected using NAD+/NADH detection Kit (WST-8 method) (Beyotime). Intracellular adenosine levels were detected using adenosine assay Kit (Abcam).
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