title: Identification of deacetylase substrates with the biotin switch approach.
abstract: 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.
authors.affiliation: Duke Proteomics Core Facility, Institute for Genome Sciences and Policy, Duke University Medical Center, Durham, NC, USA.
authors.forename: J Will
authors.initials: JW
authors.lastname: Thompson
authors.forename: Alex
authors.initials: A
authors.lastname: Robeson
authors.forename: Joshua L
authors.initials: JL
authors.lastname: Andersen
mesh_terms: D000107:Acetylation; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D000915:Antibody Affinity; D001710:Biotin / Q000378:metabolism*; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid / Q000379:methods*; D047468:Immunoprecipitation / Q000379:methods*; D008239:Lysine / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014982:Xenopus laevis / Q000378:metabolism*
chemical_list: D000906:Antibodies; D010446:Peptide Fragments; D011506:Proteins; D001710:Biotin; D008239:Lysine