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. GarciaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1977)


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.

Key words

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



Funding support from NIH grants 2T32CA009140-41A1, R01GM110174, R01AI118891, P01CA196539, and T32 GM071399 is gratefully acknowledged.


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Peder J. Lund
    • 1
  • Yekaterina Kori
    • 1
  • Xiaolu Zhao
    • 2
  • Simone Sidoli
    • 1
  • Zuo-Fei Yuan
    • 1
  • Benjamin A. Garcia
    • 1
    Email author
  1. 1.Department of Biochemistry and Biophysics, Perelman School of Medicine, Epigenetics InstituteUniversity of PennsylvaniaPhiladelphiaUSA
  2. 2.Hubei Key Laboratory of Cell Homeostasis, College of Life SciencesWuhan UniversityWuhanChina

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