SIRT1 top 40 hits: Use of one-bead, one-compound acetyl-peptide libraries and quantum dots to probe deacetylase specificity
SIRT1 top 40 hits: Use of one-bead, one-compound acetyl-peptide libraries and quantum dots to probe deacetylase specificity
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DOI：
10.1021/bi052015l
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发表时间：
2006-01-10
期刊：
影响因子：
2.9
通讯作者：
Denu, JM
中科院分区：
文献类型：
--
作者：
Garske, AL;Denu, JM
利用单珠单化合物（OBOC）乙酰肽库，结合量子点标记策略和自动化珠分选技术，建立了一种高通量测定脱乙酰酶底物特异性的新方法。围绕中心C-氨基乙酰化赖氨酸构建了104907个独特序列的5-mer OBOC肽文库。使用人NAD（+）依赖性脱乙酰酶SIRT 1筛选文库中最有效的脱乙酰化肽序列。通过SIRT 1优先脱乙酰化的珠被生物素化并用链霉亲和素包被的量子点标记。荧光珠分选后，通过质谱法对前39个最亮的珠进行测序。随机选择命中和nonhit序列的溶液中脱乙酰酶测定显示，命中与催化活性增加多达20倍。我们发现，SIRT 1可以区分肽底物的上下文依赖性的方式。
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 c-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.