title: Engineered bromodomains to explore the acetylproteome.
abstract: MS-based analysis of the acetylproteome has highlighted a role for acetylation in a wide array of biological processes including gene regulation, metabolism, and cellular signaling. To date, anti-acetyllysine antibodies have been used as the predominant affinity reagent for enrichment of acetyllysine-containing peptides and proteins; however, these reagents suffer from high nonspecific binding and lot-to-lot variability. Bromodomains represent potential affinity reagents for acetylated proteins and peptides, given their natural role in recognition of acetylated sequence motifs in vivo. To evaluate their efficacy, we generated recombinant proteins representing all known yeast bromodomains. Bromodomain specificity for acetylated peptides was determined using degenerate peptide arrays, leading to the observation that different bromodomains display a wide array of binding specificities. Despite their relatively weak affinity, we demonstrate the ability of selected bromodomains to enrich acetylated peptides from a complex biological mixture prior to mass spectrometric analysis. Finally, we demonstrate a method for improving the utility of bromodomain enrichment for MS through engineering novel affinity reagents using combinatorial tandem bromodomain pairs.
authors: Department of Biological Engineering, MIT, Cambridge, MA, USA; David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA.
authors: Bryan D
authors: BD
authors: Bryson
authors: Amanda M
authors: AM
authors: Del Rosario
authors: Jonathan S
authors: JS
authors: Gootenberg
authors: Michael B
authors: MB
authors: Yaffe
authors: Forest M
authors: FM
authors: White
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006657:Histones / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000032:analysis* / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000092468:Bromodomain Containing Proteins
chemical_list: D004268:DNA-Binding Proteins; D006657:Histones; D008239:Lysine; D010455:Peptides; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C091342:BDF1 protein, S cerevisiae; D000092468:Bromodomain Containing Proteins; C103337:RSC complex, S cerevisiae
keywords: Affinity reagent; Bromodomain; Lysine acetylation; Posttranslational modification; Protein engineering; Technology
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