Phosphoproteomic approach to characterize protein mono- and poly(ADP-ribosyl)ation sites from cells.
Phosphoproteomic approach to characterize protein mono- and poly(ADP-ribosyl)ation sites from cells.
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DOI：
10.1021/pr401032q
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发表时间：
2014-08-01
影响因子：
4.4
通讯作者：
Leung, Anthony K. L.
中科院分区：
文献类型：
--
作者：
Daniels, Casey M.;Ong, Shao-En;Leung, Anthony K. L.
关键词：
聚（adp -核糖）或PAR是一种细胞聚合物，通过其作为翻译后修饰、信号分子和支架元件的作用，参与DNA/RNA代谢、细胞死亡和细胞应激反应。PAR是由被称为聚（adp -核糖）聚合酶（parp）的蛋白质家族合成的，parp将PAR聚合物附着在底物蛋白质的各种氨基酸上。这些聚合物的性质（大的、带电的、不均匀的、碱基不稳定的）使得这些附着位点难以用质谱法研究。在这里，我们提出了一个新的管道，允许通过磷酸二酯酶处理的adp核糖或磷酸核糖的酶产物来识别单（adp -核糖基）化和多（adp -核糖基）化位点。这种方法的强大之处在于磷酸核糖的富集潜力，我们发现，当使用中性缓冲液（允许保留碱基不稳定的附着位点）进行洗脱时，磷酸蛋白质组学技术可以富集磷酸核糖。通过对PARP-1体外自修饰位点和全细胞内源性adp核糖基化位点的鉴定，我们发现除了已知的酸性修饰位点外，adp核糖还可以存在于邻近的氨基酸残基以及赖氨酸和精氨酸上。该技术的普遍性使我们能够证明，通过大结构域富集adp -核糖基化的蛋白质会导致对与谷氨酸结合的adp -核糖修饰的偏见，这表明大结构域正在去除或选择这些不同的蛋白质附着。最终，这里提出的富集管道提供了一种通用的方法来表征单和多（adp -核糖基）蛋白组。
Poly(ADP-ribose), or PAR, is a cellular polymer implicated in DNA/RNA metabolism, cell death, and cellular stress response via its role as a post-translational modification, signaling molecule, and scaffolding element. PAR is synthesized by a family of proteins known as poly(ADP-ribose) polymerases, or PARPs, which attach PAR polymers to various amino acids of substrate proteins. The nature of these polymers (large, charged, heterogeneous, base-labile) has made these attachment sites difficult to study by mass spectrometry. Here we propose a new pipeline that allows for the identification of mono(ADP-ribosyl)ation and poly(ADP-ribosyl)ation sites via the enzymatic product of phosphodiesterase-treated ADP-ribose, or phospho(ribose). The power of this method lies in the enrichment potential of phospho(ribose), which we show to be enriched by phosphoproteomic techniques when a neutral buffer, which allows for retention of the base-labile attachment site, is used for elution. Through the identification of PARP-1 in vitro automodification sites as well as endogenous ADP-ribosylation sites from whole cells, we have shown that ADP-ribose can exist on adjacent amino acid residues as well as both lysine and arginine in addition to known acidic modification sites. The universality of this technique has allowed us to show that enrichment of ADP-ribosylated proteins by macrodomain leads to a bias against ADP-ribose modifications conjugated to glutamic acids, suggesting that the macrodomain is either removing or selecting against these distinct protein attachments. Ultimately, the enrichment pipeline presented here offers a universal approach for characterizing the mono- and poly(ADP-ribosyl)ated proteome.
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影响因子：
4.4
作者：
Chapman, John D.;Gagne, Jean-Philippe;Goodett, David R.
通讯作者：
Goodett, David R.
影响因子：
14.9
作者：
Gagné JP;Pic E;Isabelle M;Krietsch J;Ethier C;Paquet E;Kelly I;Boutin M;Moon KM;Foster LJ;Poirier GG
通讯作者：
Poirier GG
影响因子：
64.5
作者：
Huttlin EL;Jedrychowski MP;Elias JE;Goswami T;Rad R;Beausoleil SA;Villén J;Haas W;Sowa ME;Gygi SP
通讯作者：
Gygi SP
DOI：
10.1016/0167-4838(91)99007-f
发表时间：
1991-06-24
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
DESMARAIS, Y;MENARD, L;POIRIER, GG
通讯作者：
POIRIER, GG
影响因子：
4
作者：
Arnold, J;Grune, T
通讯作者：
Grune, T