Reader Domain-Based Capture
Kac-Specific Capture Reagents
Kacs are recognized and bound in vivo by bromodomains, via four a-helices linked by loop regions, which provide a specific binding site. A systematic evaluation of bromodomain efficacy as affinity capture reagents has been undertaken for Saccharomyces cerevisiae bromodomains . The different bromodomains were demonstrated to have variable binding specificity, indicating pan lysine capture potential in a manner reflecting the natural diversity of acetylation sites. Proof of principle was demonstrated using immobilized GST-His6-tagged BDF1-B fusion protein for affinity capture followed by MS analysis and found to have enrichment efficiency similar to that of pan-acetyl antibodies. Furthermore, coupling of pairs of bromodomains enhanced capture efficiency as demonstrated for capture of histone lysine-acetylated peptides using both bromodomains of BDF-1 protein . This strategy represents a potential step change in analysis of lysine acetylation, providing reagents that can be engineered to increase their selectivity and affinity toward acetylated sequences .