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Quantitative Analysis of the Sirt5-Regulated Lysine Succinylation Proteome in Mammalian Cells

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Quantitative Proteomics by Mass Spectrometry

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1410))

Abstract

Lysine (Lys) succinylation is a recently discovered protein posttranslational modification pathway that is evolutionarily conserved from bacteria to mammals. It is regulated by Sirt5, a member of the class III histone deacetylases (HDACs) or the Sirtuins. Recent studies demonstrated that Lys succinylation and Sirt5 are involved in diverse cellular metabolic processes including urea cycle, ammonia transfer, and glucose metabolism. In this chapter, we describe the general protocol to identify Sirt5-regulated Lys succinylation substrates and a computational method to calculate the absolute modification stoichiometries of Lys succinylation sites. The strategy employs Stable Isotope Labeling of Amino acid in Cell culture (SILAC) and the immunoaffinity enrichment of Lys succinylated peptides to identify the Lys succinylation sites that are significantly upregulated in Sirt5 knockout mouse embryonic fibroblast cells.

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Acknowledgements

The author would like to thank Drs. Yingming Zhao, Timothy Griffin, and Minjia Tan for their critical reading of the manuscript. The work is supported by Minnesota Medical Foundation Grant and Startup funding from the University of Minnesota at Twin Cities.

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Authors and Affiliations

  1. Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321Church Street S.E.,, Minneapolis, MN, 55455, USA

    Yue Chen

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  1. Yue Chen
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Correspondence to Yue Chen .

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Editors and Affiliations

  1. NIDDK, National Institutes of Health, BETHESDA, Maryland, USA

    Salvatore Sechi

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Chen, Y. (2016). Quantitative Analysis of the Sirt5-Regulated Lysine Succinylation Proteome in Mammalian Cells. In: Sechi, S. (eds) Quantitative Proteomics by Mass Spectrometry. Methods in Molecular Biology, vol 1410. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3524-6_2

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  • DOI: https://doi.org/10.1007/978-1-4939-3524-6_2

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3522-2

  • Online ISBN: 978-1-4939-3524-6

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