Abstract：
Multifaceted interrogation of the proteome deepens the system-wide understanding of biological systems; however, mapping the redox changes in the proteome has so far been significantly more challenging than expression and solubility/stability analyses. Here, the first high-throughput redox proteomics approach integrated with expression analysis (REX) is devised and combined with the Proteome Integral Solubility Alteration (PISA) assay. The whole PISA-REX experiment with up to four biological replicates can be multiplexed into a single tandem mass tag TMTpro set. For benchmarking this compact tool, HCT116 cells treated with auranofin are analyzed, showing great improvement compared with previous studies. PISA-REX is then applied to study proteome remodeling upon stimulation of human monocytes by interferon α (IFN-α). Applying this tool to study the proteome changes in plasmacytoid dendritic cells (pDCs) isolated from wild-type versus Ncf1-mutant mice treated with interferon α, shows that NCF1 deficiency enhances the STAT1 pathway and modulates the expression, solubility, and redox state of interferon-induced proteins. Providing comprehensive multifaceted information on the proteome, the compact PISA-REX has the potential to become an industry standard in proteomics and to open new windows into the biology of health and disease.
摘要：
对蛋白质组的多方面询问加深了对生物系统的全系统理解；然而，迄今为止,蛋白质组的氧化还原变化比表达和溶解度/稳定性分析更具挑战性.这里,设计了第一个与表达分析（REX）整合的高通量氧化还原蛋白质组学方法，并将其与蛋白质组积分溶解度改变（PISA）测定相结合。具有多达四个生物学重复的整个PISA-REX实验可以多路复用成单个串联质量标签TMTpro组。为了对这个紧凑的工具进行基准测试，分析了用金诺芬处理的HCT116细胞，与以前的研究相比有很大的改进。然后将PISA-REX用于研究干扰素α（IFN-α）刺激人单核细胞后的蛋白质组重塑。应用该工具研究从用干扰素α处理的野生型与Ncf1突变小鼠中分离的浆细胞样树突状细胞（pDC）的蛋白质组变化，显示NCF1缺乏增强STAT1途径并调节表达，溶解度，和干扰素诱导蛋白的氧化还原状态。提供关于蛋白质组的全面多方面的信息，紧凑型PISA-REX有可能成为蛋白质组学的行业标准，并为健康和疾病生物学打开新的窗口。
