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使用代谢标记和质谱法测量衰老和非分裂培养细胞中的蛋白质周转率

JoVE Journal
Biochimica

This content is Free Access.

JoVE Journal Biochimica

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

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使用代谢标记和质谱法测量衰老和非分裂培养细胞中的蛋白质周转率

DOI:

10.3791/63835-v

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08:52 min

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April 06, 2022

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Matthew Payea, Myriam Gorospe, Nathan Basisty

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

Capitoli

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

Traduzione automatica

该协议描述了使用脉冲SILAC，非靶向质谱分析和简化的蛋白质半衰期计算来标记衰老和非分裂细胞的代谢标记的工作流程。

Tags

Keywords: Protein Turnover Senescent Cells Non-dividing Cells Metabolic Labeling Mass Spectrometry SILAC Protein Homeostasis Cellular Stress Aging Neurodegenerative Disease LC-MS Trypsin Urea Ammonium Bicarbonate Dithiothreitol Iodoacetamide

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