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Identification of deacetylase substrates with the biotin switch approach.

Publication ,  Journal Article
Thompson, JW; Robeson, A; Andersen, JL
Published in: Methods Mol Biol
2013

The identification of lysine-acetylated proteins and deacetylase substrates has primarily relied on protein immune-affinity techniques with antibodies that recognize acetylated lysine residues (Kac antibodies). While these antibody-based techniques are continuously improving, they can be limited by the narrow and many times unknown epitope specificity of Kac antibodies. An alternative approach is the biotin switch capture of deacetylated proteins. Similar in part to other biotin switch methodologies, this technique relies on the blocking of native lysine residues and removal of the modification of interest in vitro, after which the newly deacetylated proteins can be captured and identified by mass spectrometry (MS). In this chapter, we cover the essential steps of the procedure, highlight key points in the assay to reduce false positive protein identification, and discuss the quantitative MS methods useful for identifying the captured deacetylase substrates. We also discuss potential strategies and future improvements to overcome current limitations of the assay.

Duke Scholars

Published In

Methods Mol Biol

DOI

EISSN

1940-6029

Publication Date

2013

Volume

1077

Start / End Page

133 / 148

Location

United States

Related Subject Headings

  • Xenopus laevis
  • Tandem Mass Spectrometry
  • Substrate Specificity
  • Proteins
  • Protein Processing, Post-Translational
  • Peptide Fragments
  • Lysine
  • Immunoprecipitation
  • Developmental Biology
  • Chromatography, Liquid
 

Citation

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Thompson, J. W., Robeson, A., & Andersen, J. L. (2013). Identification of deacetylase substrates with the biotin switch approach. Methods Mol Biol, 1077, 133–148. https://doi.org/10.1007/978-1-62703-637-5_9
Thompson, J Will, Alex Robeson, and Joshua L. Andersen. “Identification of deacetylase substrates with the biotin switch approach.Methods Mol Biol 1077 (2013): 133–48. https://doi.org/10.1007/978-1-62703-637-5_9.
Thompson JW, Robeson A, Andersen JL. Identification of deacetylase substrates with the biotin switch approach. Methods Mol Biol. 2013;1077:133–48.
Thompson, J. Will, et al. “Identification of deacetylase substrates with the biotin switch approach.Methods Mol Biol, vol. 1077, 2013, pp. 133–48. Pubmed, doi:10.1007/978-1-62703-637-5_9.
Thompson JW, Robeson A, Andersen JL. Identification of deacetylase substrates with the biotin switch approach. Methods Mol Biol. 2013;1077:133–148.

Published In

Methods Mol Biol

DOI

EISSN

1940-6029

Publication Date

2013

Volume

1077

Start / End Page

133 / 148

Location

United States

Related Subject Headings

  • Xenopus laevis
  • Tandem Mass Spectrometry
  • Substrate Specificity
  • Proteins
  • Protein Processing, Post-Translational
  • Peptide Fragments
  • Lysine
  • Immunoprecipitation
  • Developmental Biology
  • Chromatography, Liquid