Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Vienna, Austria.
Department of Tumor Biology, CCRI, Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria.
J Proteomics. 2017 Jan 30;152:161-171. doi: 10.1016/j.jprot.2016.11.008. Epub 2016 Nov 13.
Peripheral blood mononuclear cells are important players in immune regulation relying on a complex network of signaling pathways. In this study, we evaluated the power of label-free quantitative shotgun proteomics regarding the comprehensive characterization of signaling pathways in such primary cells by studying regulation of protein abundance, post-translational modifications and nuclear translocation events. The effects of inflammatory stimulation and the treatment of stimulated cells with dexamethasone were investigated. Therefore, a previously published dataset accessible via ProteomeXchange consisting of 6901 identified protein groups was re-evaluated. These data enabled us to comprehensively map the c-JUN, ERK5 and NF-κB signaling cascade in a semi-quantitative fashion. Without the application of any enrichment, 3775 highly confident phosphopeptides derived from 1249 proteins including 66 kinases were identified. Efficient subcellular fractionation and subsequent comparative analysis identified previously unrecognized inflammation-associated nuclear translocation events of proteins such as histone-modifying proteins, zinc finger proteins as well as transcription factors. Profound effects of inflammatory stimulation and dexamethasone treatment on histone H3 and ZFP161 localization represent novel findings and were verified by immunofluorescence. In conclusion, we demonstrate that multiple regulatory events resulting from the activity of signaling pathways can be determined out of untargeted shotgun proteomics data.
Relevant functional events such as phosphorylation and nuclear translocation of proteins were extracted from high-resolution mass spectrometry data and provided additional biological information contained in shotgun proteomics data.
外周血单核细胞是免疫调节的重要参与者，依赖于复杂的信号通路网络。在这项研究中，我们通过研究蛋白质丰度、翻译后修饰和核易位事件的调节，评估了无标记定量 shotgun 蛋白质组学在这种原代细胞中全面描述信号通路的能力。研究了炎症刺激和用地塞米松处理刺激细胞的效果。因此，我们重新评估了先前通过 ProteomeXchange 可获得的包含 6901 个鉴定蛋白组的已发表数据集。这些数据使我们能够以半定量方式全面绘制 c-JUN、ERK5 和 NF-κB 信号级联。在没有应用任何富集的情况下，从包括 66 种激酶在内的 1249 种蛋白质中鉴定出 3775 个高度可信的磷酸肽。有效的亚细胞分级分离和随后的比较分析确定了以前未被识别的与炎症相关的核易位事件，如组蛋白修饰蛋白、锌指蛋白以及转录因子。炎症刺激和地塞米松处理对组蛋白 H3 和 ZFP161 定位的深刻影响代表了新的发现，并通过免疫荧光验证。总之，我们证明了可以从无靶向 shotgun 蛋白质组学数据中确定信号通路活性产生的多种调节事件。
从高分辨率质谱数据中提取了相关的功能事件，如蛋白质的磷酸化和核易位，并提供了 shotgun 蛋白质组学数据中包含的其他生物学信息。
