Web Release Date: December 13,
SIRT1 Top 40 Hits: Use of One-Bead, One-Compound Acetyl-Peptide Libraries
and Quantum Dots to Probe Deacetylase Specificity
and
Departments of Biomolecular Chemistry and Chemistry, University of Wisconsin, Madison, Wisconsin 53706
Received October 4, 2005
Revised Manuscript Received November 9, 2005
Abstract:
A novel, high-throughput method for determining deacetylase substrate specificity was
developed using a one-bead, one-compound (OBOC) acetyl-peptide library with a quantum dot tagging
strategy and automated bead-sorting. A 5-mer OBOC peptide library of 104 907 unique sequences was
constructed around a central 
-amino acetylated lysine. The library was screened using the human NAD+-dependent deacetylase SIRT1 for the most efficiently deacetylated peptide sequences. Beads preferentially
deacetylated by SIRT1 were biotinylated and labeled with streptavidin-coated quantum dots. After
fluorescent bead-sorting, the top 39 brightest beads were sequenced by mass spectrometry. In-solution
deacetylase assays on randomly chosen hit and nonhit sequences revealed that hits correlated with increased
catalytic activity by as much as 20-fold. We found that SIRT1 can discriminate peptide substrates in a
context-dependent fashion.
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