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Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems
JoVE Journal
Bioengineering
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JoVE Journal Bioengineering
Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

DOI:
10.3791/62852-v

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14:42 min

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September 23, 2021

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, , ,
1Graduate School of Genome Science & Technology,University of Tennessee Knoxville, 2Bredesen Center for Interdisciplinary Research,University of Tennessee Knoxville, 3Biosciences Division,Oak Ridge National Laboratory

Chapters

  • 00:04Introduction
  • 01:08Starting, Stopping, and Processing Time Course CFME Reactions for HPLC-RID Quantification
  • 03:18Creating a Sequence Table for Autosampling and Start the HPLC-RID System for Data Acquisition
  • 04:54Extracting and Analyzing Data Post-Run
  • 07:20Starting, Stopping, and Processing Time Course Isotope Tracing CFME Reactions for LC-MS/MS Quantification
  • 09:20Setting Up a Run Sequence and Starting the LC-MS/MS Run
  • 10:16Calculating Negative Mode Masses of 13C-Labeled Glucose-Derived Metabolites and Searching for the m/z Features of These Analytes in Filtered Data
  • 11:45Results: Metabolite Profiling in Lysate-based Cell-free Systems
  • 13:54Conclusion

Summary

Automatic Translation

Automatic Translation

The protocols describe high-performance liquid chromatography methods coupled to refractive index or mass spectrometric detection for studying metabolic reactions in complex lysate-based cell-free systems.

Tags

Keywords: Liquid ChromatographyRefractive Index DetectionMass SpectrometryMetabolite ProfilingCell-free SystemsCarbon MetabolismCentral MetabolitesLysate-basedHPLCSample PreparationAcidificationFiltration

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