SIRT1 top 40 hits: use of one-bead, one-compound acetyl-peptide libraries and quantum dots to probe deacetylase specificity.
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 epsilon-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.
Garske
Adam L
AL
Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Denu
John M
JM
D000107:Acetylation; D000596:Amino Acids / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D002384:Catalysis; D006655:Histone Deacetylases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D009243:NAD / Q000378:metabolism; D019151:Peptide Library; D045663:Quantum Dots; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D056564:Sirtuin 1; D037761:Sirtuins / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D019809:Streptavidin / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D000596:Amino Acids; D019151:Peptide Library; D009243:NAD; D001710:Biotin; D019809:Streptavidin; C447939:SIRT1 protein, human; D056564:Sirtuin 1; D037761:Sirtuins; D006655:Histone Deacetylases; D008239:Lysine