Title | Engineered bromodomains to explore the acetylproteome. |
Publication Type | Journal Article |
Year of Publication | 2015 |
Authors | Bryson, BD, Del Rosario, AM, Gootenberg, JS, Yaffe, MB, White, FM |
Journal | Proteomics |
Volume | 15 |
Issue | 9 |
Pagination | 1470-5 |
Date Published | 2015 May |
ISSN | 1615-9861 |
Keywords | Acetylation, Amino Acid Sequence, DNA-Binding Proteins, Histones, Humans, Lysine, Molecular Sequence Data, Peptides, Protein Engineering, Protein Processing, Post-Translational, Protein Structure, Tertiary, Proteomics, Recombinant Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Tandem Mass Spectrometry, Transcription Factors |
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. |
DOI | 10.1002/pmic.201400401 |
Alternate Journal | Proteomics |
PubMed ID | 25641834 |
PubMed Central ID | PMC4535431 |
Grant List | P30 CA014051 / CA / NCI NIH HHS / United States P30 ES002109 / ES / NIEHS NIH HHS / United States P30CA14051 / CA / NCI NIH HHS / United States R01 CA096504 / CA / NCI NIH HHS / United States R01 CA118705 / CA / NCI NIH HHS / United States R01 ES015339 / ES / NIEHS NIH HHS / United States R01 GM104047 / GM / NIGMS NIH HHS / United States R01 GM104047 / GM / NIGMS NIH HHS / United States R01CA118705 / CA / NCI NIH HHS / United States R01ES015339 / ES / NIEHS NIH HHS / United States R24 DK090963 / DK / NIDDK NIH HHS / United States R24DK090963 / DK / NIDDK NIH HHS / United States U24 CA159988 / CA / NCI NIH HHS / United States U24CA159988 / CA / NCI NIH HHS / United States U54 CA112967 / CA / NCI NIH HHS / United States U54CA112967 / CA / NCI NIH HHS / United States |