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Abstract：
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.
摘要：
我们已经使用质谱（MS）来表征来自人血的脂多糖（LPS）刺激的巨噬细胞中的蛋白质信号，人THP1细胞，小鼠骨髓，和小鼠Raw264.7细胞。蛋白质ADP-核糖基化被截断为磷酸核糖，允许与磷酸肽一起富集和鉴定所得的磷酸化肽。大小排阻色谱-MS（SEC-MS）用于按大小分离蛋白形式；然后通过加权相关网络分析（WGCNA）鉴定蛋白质复合物，基于它们在刺激后进入或离开SEC级分的相关运动，提出了一种不依赖已建立数据库的SEC-MS分析方法。我们强调了两个感兴趣的模块：一个与凋亡信号调节激酶（ASK）信号体相连，另一个包含聚（ADP-核糖）聚合酶9（PARP9）。最后,PARP抑制被用来扰乱表征的系统，证明了ADP-核糖基化对全球相互作用组的重要性。所有翻译后修饰（PTM）和相互作用组数据已汇总到6729种蛋白质的meta数据库中，在2905蛋白上表征为ADP-核糖基化,在2669蛋白上表征为磷酸化。这个名为MAPCD的数据库，对于巨噬细胞ADP核糖基化，磷酸化,和复杂动力学-作为研究ADP-核糖基之间串扰的宝贵资源，磷酸化蛋白质组，和互动。
