Identification of deacetylase substrates with the biotin switch approach.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Thompson, JW; Robeson, A; Andersen, JL

Published Date

  • 2013

Published In

Volume / Issue

  • 1077 /

Start / End Page

  • 133 - 148

PubMed ID

  • 24014404

Pubmed Central ID

  • 24014404

Electronic International Standard Serial Number (EISSN)

  • 1940-6029

Digital Object Identifier (DOI)

  • 10.1007/978-1-62703-637-5_9

Language

  • eng

Conference Location

  • United States