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Site-Specific Lysine Acetylation Stoichiometry Across Subcellular Compartments

Part of the Methods in Molecular Biology book series (MIMB,volume 1983)

Abstract

Posttranslational modifications of proteins control many complex biological processes, including genome expression, chromatin dynamics, metabolism, and cell division through a language of chemical modifications. Improvements in mass spectrometry-based proteomics have demonstrated protein acetylation is a widespread and dynamic modification in the cell; however, many questions remain on the regulation and downstream effects, and an assessment of the overall acetylation stoichiometry is needed. In this chapter, we describe the determination of acetylation stoichiometry using data-independent acquisition mass spectrometry to expand the number of acetylation sites quantified. However, the increased depth of data-independent acquisition is limited by the spectral library used to deconvolute fragmentation spectra. We describe a powerful approach of subcellular fractionation in conjunction with offline prefractionation to increase the depth of the spectral library. This deep interrogation of subcellular compartments provides essential insights into the compartment-specific regulation and downstream functions of protein acetylation.

Keywords

  • Protein acetylation
  • Acetylation
  • Mitochondria
  • Chromatin
  • Stoichiometry
  • Posttranslational modification
  • Mass spectrometry
  • Proteomics

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Acknowledgments

We would like to thank Ian Lienert, Tejas Gandhi, Oliver Bernhardt, Lukas Reiter from Biognosys for the development of the software to generate the in silico labeled spectral library and analyze DIA MS data. This work was supported by GM65386 to J.M.D. and by NIH National Research Service Award T32 GM007215 to (A.L. and J.B.)

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Lindahl, A.J., Lawton, A.J., Baeza, J., Dowell, J.A., Denu, J.M. (2019). Site-Specific Lysine Acetylation Stoichiometry Across Subcellular Compartments. In: Brosh, Jr., R. (eds) Protein Acetylation. Methods in Molecular Biology, vol 1983. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9434-2_6

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  • DOI: https://doi.org/10.1007/978-1-4939-9434-2_6

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9433-5

  • Online ISBN: 978-1-4939-9434-2

  • eBook Packages: Springer Protocols