Isotopic labeling and quantitative proteomics of acetylation on histones and beyond

Peder J. Lund, Yekaterina Kori, Xiaolu Zhao, Simone Sidoli, Zuo Fei Yuan, Benjamin A. Garcia

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Lysine acetylation is an important posttranslational modification (PTM) that regulates the function of proteins by affecting their localization, stability, binding, and enzymatic activity. Aberrant acetylation patterns have been observed in numerous diseases, most notably cancer, which has spurred the development of potential therapeutics that target acetylation pathways. Mass spectrometry (MS) has become the most adopted tool not only for the qualitative identification of acetylation sites but also for their large-scale quantification. By using heavy isotope labeling in cell culture combined with MS, it is now possible to accurately quantify newly synthesized acetyl groups and other PTMs, allowing differentiation between dynamically regulated and steady-state modifications. Here, we describe MS-based protocols to identify acetylation sites and quantify acetylation rates on both proteins in general and in the special case of histones. In the experimental approach for the former, 13C-glucose and D3-acetate are used to metabolically label protein acetylation in cells with stable isotopes, thus allowing isotope incorporation to be tracked over time. After protein extraction and digestion, acetylated peptides are enriched via immunoprecipitation and then analyzed by MS. For histones, a similar metabolic labeling approach is performed, followed by acid extraction, derivatization with propionic anhydride, and trypsin digestion prior to MS analysis. The procedures presented may be adapted to investigate acetylation dynamics in a broad range of experimental contexts, including different cell types and stimulation conditions.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages43-70
Number of pages28
DOIs
Publication statusPublished - Jan 1 2019

Publication series

NameMethods in Molecular Biology
Volume1977
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

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Keywords

  • Acetylation dynamics
  • Epigenetics
  • Histone acetylation
  • Isotopic labeling
  • Mass spectrometry
  • Posttranslational modifications
  • Protein acetylation
  • Proteomics

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Lund, P. J., Kori, Y., Zhao, X., Sidoli, S., Yuan, Z. F., & Garcia, B. A. (2019). Isotopic labeling and quantitative proteomics of acetylation on histones and beyond. In Methods in Molecular Biology (pp. 43-70). (Methods in Molecular Biology; Vol. 1977). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-9232-4_5