Quantitative proteomics profiling of the poly(ADP-ribose)-related response to genotoxic stress.
Quantitative proteomics profiling of the poly(ADP-ribose)-related response to genotoxic stress.
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DOI：
10.1093/nar/gks486
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发表时间：
2012-09
影响因子：
14.9
通讯作者：
Poirier GG
中科院分区：
文献类型：
--
作者：
Gagné JP;Pic E;Isabelle M;Krietsch J;Ethier C;Paquet E;Kelly I;Boutin M;Moon KM;Foster LJ;Poirier GG
DNA依赖的聚(ADP-核糖)聚合酶(PARP)在DNA损伤诱导下合成一种阴离子聚(ADP-核糖)(PADPr)支架，几种蛋白质结合在该支架上，随后形成与pADPr相关的多蛋白复合体。我们使用亲和纯化方法和蛋白质组学方法相结合的方法来分离这些复合体，并评估与pADPr代谢相关的蛋白质动力学。作为第一种方法，我们开发了底物捕获策略，通过该策略，我们证明了催化失活的聚(ADP-核糖)糖水解酶(PARG)突变体可以作为生理选择性诱饵，通过其大结构域样结构域分离特定的pADPr结合蛋白。除了抗体介导的亲和纯化方法外，我们还使用pADPr大域亲和树脂回收pADPr结合蛋白及其复合体。其次，我们设计了一个时程实验，以探索烷基化DNA损伤介导的PARP激活时含有pADPr的多蛋白复合体组成的变化。基于GeLC-MS/MS分析的光谱计数聚类得到了进一步分析的补充，使用了通过相对和绝对定量等压标签(ITRAQ)的高精度定量蛋白质组学以及基于细胞培养中的氨基酸的稳定同位素标记(SILAC)的蛋白质组学。在这里，我们提供了一个有价值的资源，在多聚(ADP-核糖基)的背景下解释DNA损伤反应网络的系统生物学，并为后续研究pADPr结合蛋白候选提供基础。
Upon DNA damage induction, DNA-dependent poly(ADP-ribose) polymerases (PARPs) synthesize an anionic poly(ADP-ribose) (pADPr) scaffold to which several proteins bind with the subsequent formation of pADPr-associated multiprotein complexes. We have used a combination of affinity-purification methods and proteomics approaches to isolate these complexes and assess protein dynamics with respect to pADPr metabolism. As a first approach, we developed a substrate trapping strategy by which we demonstrate that a catalytically inactive Poly(ADP-ribose) glycohydrolase (PARG) mutant can act as a physiologically selective bait for the isolation of specific pADPr-binding proteins through its macrodomain-like domain. In addition to antibody-mediated affinity-purification methods, we used a pADPr macrodomain affinity resin to recover pADPr-binding proteins and their complexes. Second, we designed a time course experiment to explore the changes in the composition of pADPr-containing multiprotein complexes in response to alkylating DNA damage-mediated PARP activation. Spectral count clustering based on GeLC-MS/MS analysis was complemented with further analyses using high precision quantitative proteomics through isobaric tag for relative and absolute quantitation (iTRAQ)- and Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics. Here, we present a valuable resource in the interpretation of systems biology of the DNA damage response network in the context of poly(ADP-ribosyl)ation and provide a basis for subsequent investigations of pADPr-binding protein candidates.
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影响因子：
3.8
作者：
Banáth JP;Klokov D;MacPhail SH;Banuelos CA;Olive PL
通讯作者：
Olive PL
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作者：
Ahel, Ivan;Ahel, Dragana;West, Stephen C.
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West, Stephen C.
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作者：
Montes de Oca R;Shoemaker CJ;Gucek M;Cole RN;Wilson KL
通讯作者：
Wilson KL
影响因子：
14.9
作者：
Gagne, Jean-Philippe;Isabelle, Maxim;Lo, Ken Sin;Bourassa, Sylvie;Hendzel, Michael J.;Dawson, Valina L.;Dawson, Ted M.;Poirier, Guy G.
通讯作者：
Poirier, Guy G.