Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.
Institute of Medical Psychology, Faculty of Medicine, LMU, Munich, Germany.
Nat Commun. 2021 Jan 11;12(1):254. doi: 10.1038/s41467-020-20509-1.
Protein ubiquitination is involved in virtually all cellular processes. Enrichment strategies employing antibodies targeting ubiquitin-derived diGly remnants combined with mass spectrometry (MS) have enabled investigations of ubiquitin signaling at a large scale. However, so far the power of data independent acquisition (DIA) with regards to sensitivity in single run analysis and data completeness have not yet been explored. Here, we develop a sensitive workflow combining diGly antibody-based enrichment and optimized Orbitrap-based DIA with comprehensive spectral libraries together containing more than 90,000 diGly peptides. This approach identifies 35,000 diGly peptides in single measurements of proteasome inhibitor-treated cells - double the number and quantitative accuracy of data dependent acquisition. Applied to TNF signaling, the workflow comprehensively captures known sites while adding many novel ones. An in-depth, systems-wide investigation of ubiquitination across the circadian cycle uncovers hundreds of cycling ubiquitination sites and dozens of cycling ubiquitin clusters within individual membrane protein receptors and transporters, highlighting new connections between metabolism and circadian regulation.
蛋白质泛素化几乎参与所有细胞过程。采用针对泛素衍生二甘肽残基的抗体富集策略，并结合质谱（MS），已经可以大规模研究泛素信号。然而，到目前为止，关于单轮分析的灵敏度和数据完整性，数据非依赖性采集（DIA）的功能还尚未得到探索。在这里，我们开发了一种灵敏的工作流程，将二甘肽抗体富集和优化的基于 Orbitrap 的 DIA 与包含超过 90000 个二甘肽肽段的全面谱库相结合。这种方法在单次蛋白酶体抑制剂处理细胞的测量中可鉴定出 35000 个二甘肽肽段，是数据依赖采集的两倍数量和定量准确性。将该方法应用于 TNF 信号转导，该工作流程全面捕获已知的位点，同时添加了许多新的位点。对昼夜节律周期内泛素化的深入、系统的研究揭示了数百个周期性泛素化位点和数十个周期性泛素簇，存在于单个膜蛋白受体和转运体中，突显了代谢与昼夜节律调节之间的新联系。
