title: Assessment of HDACi-Induced Acetylation of Nonhistone Proteins by Mass Spectrometry.
abstract: Posttranslational acetylation of lysine residues has been discovered as multifaceted regulatory modification for various nuclear, cytoplasmic, and mitochondrial proteins. The implementation of high-resolution and high-throughput mass spectrometry (MS) approaches has led to the identification of a hitherto underappreciated, large number of acetylation sites for a broad spectrum of cellular proteins. In this chapter, we describe a comprehensive protocol for the purification of an in vivo-acetylated, ectopically expressed, FLAG-epitope tagged nonhistone protein through immunoprecipitation (IP). The protocol also covers the sample preparation by SDS-PAGE, proteolytic digestion, and the analysis by LC-ESI MS. The success of this methodology, however, strongly depends on the physico-chemical properties of the respective protein(s) and the quality of selected peptide mass spectra.
authors.affiliation: Department of Biochemistry, Center for Molecular Biomedicine (CMB), Friedrich Schiller University Jena, Hans-Knöll-Str. 2, Jena, 07745, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Wieczorek
authors.affiliation: CF Proteomics, Leibniz Institute for Age Research-Fritz Lipmann Institute (FLI), Beutenberg-Str. 11, Jena, 07745, Germany.
authors.forename: Karl-Heinz
authors.initials: KH
authors.lastname: Gührs
authors.affiliation: Department of Biochemistry, Instituteof Biochemistry and Biophysics, CMB - Center for Molecular Biomedicine, Friedrich Schiller University Jena, Hans-Knöll-Str. 2, Jena, 07745, Germany. t.heinzel@uni-jena.de.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Heinzel
mesh_terms: D000107:Acetylation; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006801:Humans; D047468:Immunoprecipitation; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014162:Transfection
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D056572:Histone Deacetylase Inhibitors; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C085706:FLAG peptide
keywords: FLAG-tag purification; Histone deacetylase inhibitor; Immunoprecipitation; LC ESI tandem MS; Lysine acetylation; Mass spectrometry analysis; Posttranslational modification