Center for Interdisciplinary Cardiovascular Sciences, Division of Cardiovascular Medicine, Department of Medicine , Brigham Women's Hospital, Harvard Medical School , Boston , Massachusetts 02115 , United States.
Department of Molecular Mechanisms of Disease , University of Zurich , 8057 Zurich , Switzerland.
J Proteome Res. 2019 Apr 5;18(4):1607-1622. doi: 10.1021/acs.jproteome.8b00895. Epub 2019 Mar 21.
ADP-ribosylation is a post-translational modification that, until recently, has remained elusive to study at the cellular level. Previously dependent on radioactive tracers to identify ADP-ribosylation targets, several advances in mass spectrometric workflows now permit global identification of ADP-ribosylated substrates. In this study, we capitalized on two ADP-ribosylation enrichment strategies, and multiple activation methods performed on the Orbitrap Fusion Lumos, to identify IFN-γ-induced ADP-ribosylation substrates in macrophages. The ADP-ribosyl binding protein, Af1521, was used to enrich ADP-ribosylated peptides, and the antipoly-ADP-ribosyl antibody, 10H, was used to enrich ADP-ribosylated proteins. ADP-ribosyl-specific mass spectra were further enriched by an ADP-ribose product ion triggered EThcD and HCD activation strategy, in combination with multiple acquisitions that segmented the survey scan into smaller ranges. HCD and EThcD resulted in overlapping and unique ADP-ribosyl peptide identifications, with HCD providing more peptide identifications but EThcD providing more reliable ADP-ribosyl acceptor sites. Our acquisition strategies also resulted in the first ever characterization of ADP-ribosyl on three poly-ADP-ribose polymerases, ARTD9/PARP9, ARTD10/PARP10, and ARTD8/PARP14. IFN-γ increased the ADP-ribosylation status of ARTD9/PARP9, ARTD8/PARP14, and proteins involved in RNA processes. This study therefore summarizes specific molecular pathways at the intersection of IFN-γ and ADP-ribosylation signaling pathways.
ADP-核糖基化是一种翻译后修饰，直到最近，它在细胞水平上的研究仍然难以捉摸。以前依赖放射性示踪剂来鉴定 ADP-核糖基化的靶标，现在质谱工作流程的几项进展现在允许全局鉴定 ADP-核糖基化的底物。在这项研究中，我们利用了两种 ADP-核糖基化富集策略，以及在 Orbitrap Fusion Lumos 上进行的多种激活方法，来鉴定巨噬细胞中 IFN-γ诱导的 ADP-核糖基化底物。ADP-核糖基结合蛋白 Af1521 用于富集 ADP-核糖基化肽，抗聚 ADP-核糖基抗体 10H 用于富集 ADP-核糖基化蛋白。ADP-核糖基特异性质谱通过 ADP-核糖产物离子触发的 EThcD 和 HCD 激活策略进一步富集，结合将总扫描分成更小范围的多次采集。HCD 和 EThcD 导致重叠和独特的 ADP-核糖基肽鉴定，HCD 提供更多的肽鉴定，但 EThcD 提供更可靠的 ADP-核糖基受体位点。我们的采集策略还首次表征了三个聚 ADP-核糖聚合酶 ARTD9/PARP9、ARTD10/PARP10 和 ARTD8/PARP14 上的 ADP-核糖基化。IFN-γ 增加了 ARTD9/PARP9、ARTD8/PARP14 和参与 RNA 过程的蛋白质的 ADP-核糖基化状态。因此，本研究总结了 IFN-γ 和 ADP-核糖基化信号通路交汇点的特定分子途径。
