Mapping PARP-1 Auto-ADP-ribosylation Sites by Liquid Chromatography-Tandem Mass Spectrometry
Mapping PARP-1 Auto-ADP-ribosylation Sites by Liquid Chromatography-Tandem Mass Spectrometry
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DOI：
10.1021/pr301219h
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发表时间：
2013-04-01
影响因子：
4.4
通讯作者：
Goodett, David R.
中科院分区：
文献类型：
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作者：
Chapman, John D.;Gagne, Jean-Philippe;Goodett, David R.
我们证明了一种新的方法，用于识别聚（ADP-核糖）聚合酶-1（PARP-1）的自聚（ADP-核糖基）的网站，适合碰撞诱导解离（CID）串联质谱。通过采用磷酸二酯酶，以消除大部分的聚（ADP-核糖）（pADPr）的修改，我们降低了串联质谱分析的复杂性pADPr-修改胰蛋白酶肽。肽的简化核糖-5 '-磷酸形式通过CID产生串联质谱，其易于解释并能够有效定位PARP-1催化的聚（ADP-核糖基）化的确切位点。结合捕获核糖-5 '-磷酸肽的磷酸肽样富集策略，我们确定了PARP-1自修饰的8个新位点，证实了先前报道的两个位点的定位，并为两个具有模糊修饰位点分配的额外靶向肽提供了证据。由于该方法简单，该方法很容易适用于复杂样品的分析。
We demonstrate a novel method for the identification of poly(ADP-ribose) polymerase-1 (PARP-1) autopoly(ADP-ribosyl)ation sites that is suited to collision induced dissociation (CID) tandem mass spectrometry. By employing phosphodiesterase to remove the majority of the poly(ADP-ribose) (pADPr) modification, we reduce the complexity of tandem mass spectrometric analysis of pADPr-modified tryptic peptides. The simplified ribose-5'-phosphate form of the peptides produce tandem mass spectra by CID that are readily interpreted and enable effective localization of the exact sites of PARP-1-catalyzed poly(ADP-ribosyl)ation. In conjunction with a phosphopeptide-like enrichment strategy that captures the ribose-5'-phosphate peptides, we identified eight novel sites of PARP-1 automodification, confirmed the localization of two sites previously reported, and provided evidence for two additional targeted peptides with ambiguous modification site assignments. Given the simplicity of the approach, the method is readily applicable to analysis of complex samples.