Methods for characterizing protein acetylation during viral infection

Laura A. Murray, Ashton N. Combs, Pranav Rekapalli, Ileana M. Cristea

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Lysine acetylation is a prevalent posttranslational modification that acts as a regulator of protein function, subcellular localization, and interactions. A growing body of work has highlighted the importance of temporal alterations in protein acetylation during infection with a range of human viruses. It has become clear that both cellular and viral proteins are decorated by lysine acetylations, and that these modifications contribute to core host defense and virus replication processes. Further defining the extent and dynamics of protein acetylation events during the progression of an infection can provide an important new perspective on the intricate mechanisms underlying the biology and pathogenesis of virus infections. Here, we provide protocols for identifying, quantifying, and probing the regulation of lysine acetylations during viral infection. We describe the use of acetyl-lysine immunoaffinity purification and quantitative mass spectrometry for assessing the cellular acetylome at different stages of an infection. As an alternative to traditional antibody-mediated western blotting, we discuss the benefits of targeted mass spectrometry approaches for detecting and quantifying site-specific acetylations on proteins of interest. Specifically, we provide a protocol using parallel reaction monitoring (PRM). We further discuss experimental considerations that are specific to studying viral infections. Finally, we provide a brief overview of the types of assays that can be employed to characterize the function of an acetylation event in the context of infection. As a method to interrogate the regulation of acetylation, we describe the Fluor de Lys assay for monitoring the enzymatic activities of deacetylases.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
EditorsBenjamin A. Garcia
PublisherAcademic Press Inc.
Pages587-620
Number of pages34
ISBN (Print)9780128186695
DOIs
StatePublished - 2019

Publication series

NameMethods in Enzymology
Volume626
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

Keywords

  • Acetylome
  • HDAC
  • Mass spectrometry
  • PRM
  • Proteomics
  • SIRT
  • Sirtuin
  • Targeted mass spectrometry

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  • Cite this

    Murray, L. A., Combs, A. N., Rekapalli, P., & Cristea, I. M. (2019). Methods for characterizing protein acetylation during viral infection. In B. A. Garcia (Ed.), Methods in Enzymology (pp. 587-620). (Methods in Enzymology; Vol. 626). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2019.06.030