An Advanced Strategy for Comprehensive Profiling of ADP- ribosylation Sites Using Mass Spectrometry- based Proteomics
An Advanced Strategy for Comprehensive Profiling of ADP- ribosylation Sites Using Mass Spectrometry- based Proteomics
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DOI：
10.1074/mcp.tir119.001315
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发表时间：
2019-05-01
影响因子：
7
通讯作者：
Nielsen, Michael L.
中科院分区：
文献类型：
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作者：
Hendriks, Ivo A.;Larsen, Sara C.;Nielsen, Michael L.
ADP-核糖基化是一种广泛存在的翻译后修饰（PTM），在许多细胞过程中具有重要功能。在这里，我们描述了一个深入的ADP-核糖基组使用我们的Af 1521为基础的蛋白质组学方法的ADP-核糖基化位点的综合分析，通过系统地评估互补的蛋白水解diglysine和前体片段，通过应用电子转移高能碰撞解离（EThcD）和电子转移解离（ETD），分别。虽然ETD光谱产生了较高的鉴定分数，但无论使用何种蛋白酶，在ADP-核糖基化位点的鉴定和定位方面，EThcD通常被证明优于ETD上级。尽管如此，互补蛋白酶和片段化方法的倾向将ADP-核糖基化的可检测库扩展到前所未有的深度。这种对受到DNA损伤的HeLa细胞中ADP-核糖基组的全系统分析揭示了> 11，000个独特的ADP-核糖基化肽，映射到> 7，000个ADP-核糖基化位点，总共修饰了超过三分之一的人类核蛋白质组，并突出了这种PTM的广泛范围。高分辨率的MS/ MS光谱能够识别几十种伴随ADP核糖基化和磷酸化修饰的蛋白质，揭示了组蛋白上相当程度的串扰。ADP-核糖基化被确信地定位于各种氨基酸残基类型，包括较少修饰的残基，其中数百个ADP-核糖基化位点精确定位于组氨酸、精氨酸和酪氨酸残基。功能富集分析表明，这些特定残基类型的修饰是以空间方式定向的，酪氨酸ADP核糖基化连接到核糖体，精氨酸ADP核糖基化连接到内质网，组氨酸ADP核糖基化连接到核糖体。Molecular & Cellular Proteomics 18：1010-1026，2019. DOI：10.1074/mcp.TIR119.001315。
ADP-ribosylation is a widespread post-translational modification (PTM) with crucial functions in many cellular processes. Here, we describe an in-depth ADP-ribosylome using our Af1521-based proteomics methodology for comprehensive profiling of ADP-ribosylation sites, by systematically assessing complementary proteolytic digestions and precursor fragmentation through application of electron-transfer higher-energy collisional dissociation (EThcD) and electron transfer dissociation (ETD), respectively. Although ETD spectra yielded higher identification scores, EThcD generally proved superior to ETD in identification and localization of ADP-ribosylation sites regardless of protease employed. Notwithstanding, the propensities of complementary proteases and fragmentation methods expanded the detectable repertoire of ADP-ribosylation to an unprecedented depth. This system-wide profiling of the ADP-ribosylome in HeLa cells subjected to DNA damage uncovered > 11,000 unique ADP-ribosylated peptides mapping to > 7,000 ADP-ribosylation sites, in total modifying over one-third of the human nuclear proteome and highlighting the vast scope of this PTM. Highresolution MS/ MS spectra enabled identification of dozens of proteins concomitantly modified by ADP-ribosylation and phosphorylation, revealing a considerable degree of crosstalk on histones. ADP-ribosylation was confidently localized to various amino acid residue types, including less abundantly modified residues, with hundreds of ADP-ribosylation sites pinpointed on histidine, arginine, and tyrosine residues. Functional enrichment analysis suggested modification of these specific residue types is directed in a spatial manner, with tyrosine ADPribosylation linked to the ribosome, arginine ADP-ribosylation linked to the endoplasmic reticulum, and histidine ADPribosylation linked to the mitochondrion. Molecular & Cellular Proteomics 18: 1010-1026, 2019. DOI: 10.1074/mcp.TIR119.001315.