A practical guide for analysis of histone post-translational modifications by mass spectrometry: Best practices and pitfalls

Methods. 2020 Dec 1:184:53-60. doi: 10.1016/j.ymeth.2019.12.001. Epub 2019 Dec 7.


Advances in mass spectrometry (MS) have revolutionized the ability to measure global changes in histone post-translational modifications (PTMs). The method routinely quantifies over 60 modification states in a single sample, far exceeding the capabilities of traditional western blotting. Thus, MS-based histone analysis has become an increasingly popular tool for understanding how genetic and environmental factors influence epigenetic states. However, histone proteomics experiments exhibit unique challenges, such as batch-to-batch reproducibility, accurate peak integration, and noisy data. Here, we discuss the steps of histone PTM analysis, from sample preparation and peak integration to data analysis and validation. We outline a set of best practices for ensuring data quality, accurate normalization, and robust statistics. Using these practices, we quantify histone modifications in 5 human cell lines, revealing that each cell line exhibits a unique epigenetic signature. We also provide a reproducible workflow for histone PTM analysis in the form of an R script, which is freely available at https://github.com/DenuLab/HistoneAnalysisWorkflow.

Keywords: Acetylation; Data analysis; Data visualization; Histone post-translational modification (PTM); Mass spectrometry; Methylation.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Data Interpretation, Statistical
  • Guidelines as Topic
  • Histone Code
  • Histones / analysis*
  • Histones / metabolism
  • Humans
  • Mass Spectrometry / methods
  • Mass Spectrometry / standards*
  • Protein Processing, Post-Translational*
  • Proteomics / methods
  • Proteomics / standards*
  • Reproducibility of Results
  • Software


  • Histones