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Engineered bromodomains to explore the acetylproteome

Author(s)
Del Rosario, Amanda M; Yaffe, Michael B; Gootenberg, Jonathan S; Bryson, Bryan D.; White, Forest M.
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Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/
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Abstract
Mass spectrometry-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 non-specific 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 mass spectrometry through engineering novel affinity reagents using combinatorial tandem bromodomain pairs.
Date issued
2015-04
URI
http://hdl.handle.net/1721.1/105868
Department
Massachusetts Institute of Technology. Department of Biological Engineering; Massachusetts Institute of Technology. Department of Biology; Koch Institute for Integrative Cancer Research at MIT
Journal
PROTEOMICS
Publisher
Wiley Blackwell
Citation
Bryson, Bryan D. et al. “Engineered Bromodomains to Explore the Acetylproteome.” PROTEOMICS 15.9 (2015): 1470–1475.
Version: Author's final manuscript
ISSN
1615-9853
1615-9861

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