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Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry
JoVE Journal
Biochemistry
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JoVE Journal Biochemistry
Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

DOI:
10.3791/63835-v

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08:52 min

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April 06, 2022

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, ,
1Laboratory of Genetics and Genomics, National Institute on Aging Intramural Research Program,National Institutes of Health, 2Translational Gerontology Branch, National Institute on Aging Intramural Research Program,National Institutes of Health

Chapters

  • 00:05Introduction
  • 00:58Labeling of Cells Pulsed SILAC and Harvest of Lysates
  • 02:29In-Solution Trypsin Digestion and Sample Clean-Up with Solid-Phase Extraction (SPE)
  • 04:15Data-Dependent Acquisition Mass Spectrometry Analysis and Calculation of Protein Half-Lives
  • 06:31Results: Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells Using pSILAC Mass Spectrometry
  • 08:13Conclusion

Summary

Automatic Translation

Automatic Translation

This protocol describes the workflow for metabolic labeling of senescent and non-dividing cells with pulsed SILAC, untargeted mass spectrometry analysis, and a streamlined calculation of protein half-lives.

Tags

Keywords: Protein TurnoverSenescent CellsNon-dividing CellsMetabolic LabelingMass SpectrometrySILACProtein HomeostasisCellular StressAgingNeurodegenerative DiseaseLC-MSTrypsinUreaAmmonium BicarbonateDithiothreitolIodoacetamide

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