We have used mass spectrometry (MS) to characterize protein signaling in lipopolysaccharide (LPS)-stimulated macrophages from human blood, human THP1 cells, mouse bone marrow, and mouse Raw264.7 cells. Protein ADP-ribosylation was truncated down to phosphoribose, allowing for enrichment and identification of the resulting phosphoribosylated peptides alongside phosphopeptides. Size exclusion chromatography-MS (SEC-MS) was used to separate proteoforms by size; protein complexes were then identified by weighted correlation network analysis (WGCNA) based on their correlated movement into or out of SEC fractions following stimulation, presenting an analysis method for SEC-MS that does not rely on established databases. We highlight two modules of interest: one linked to the apoptosis signal-regulating kinase (ASK) signalosome and the other containing poly(ADP-ribose) polymerase 9 (PARP9). Finally, PARP inhibition was used to perturb the characterized systems, demonstrating the importance of ADP-ribosylation for the global interactome. All post-translational modification (PTM) and interactome data have been aggregated into a meta-database of 6729 proteins, with ADP-ribosylation characterized on 2905 proteins and phosphorylation characterized on 2669 proteins. This database—titled MAPCD, for Macrophage ADP-ribosylation, Phosphorylation, and Complex Dynamics—serves as an invaluable resource for studying crosstalk between the ADP-ribosylome, phosphoproteome, and interactome.

中文翻译：

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LPS 激活的巨噬细胞中的动态 ADP-核糖体、磷酸化蛋白质组和相互作用组。


我们使用质谱 (MS) 来表征来自人血液、人 THP1 细胞、小鼠骨髓和小鼠 Raw264.7 细胞的脂多糖 (LPS) 刺激的巨噬细胞中的蛋白质信号传导。蛋白质 ADP 核糖基化被截断为磷酸核糖，从而可以富集和鉴定所得磷酸核糖基化肽以及磷酸肽。使用尺寸排阻色谱-MS (SEC-MS) 按尺寸分离蛋白质形式；然后，根据刺激后蛋白质复合物进出 SEC 组分的相关运动，通过加权相关网络分析 (WGCNA) 来识别蛋白质复合物，从而提出一种不依赖于已建立数据库的 SEC-MS 分析方法。我们重点介绍了两个感兴趣的模块：一个与凋亡信号调节激酶 (ASK) 信号体相关，另一个包含聚 (ADP-核糖) 聚合酶 9 (PARP9)。最后，使用 PARP 抑制来干扰表征的系统，证明 ADP-核糖基化对于整体相互作用组的重要性。所有翻译后修饰 (PTM) 和相互作用组数据均已汇总到包含 6729 个蛋白质的元数据库中，其中 2905 个蛋白质具有 ADP 核糖基化特征，2669 个蛋白质具有磷酸化特征。该数据库名为 MAPCD，代表巨噬细胞A DP-核糖基化、磷酸化和复杂动力学，是研究 ADP-核糖体、磷酸蛋白质组和相互作用组之间串扰的宝贵资源。