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。