Data-independent acquisition method for ubiquitinome analysis reveals regulation of circadian biology.
Data-independent acquisition method for ubiquitinome analysis reveals regulation of circadian biology.
复制标题
DOI：
10.1038/s41467-020-20509-1
复制
发表时间：
2021-01-11
影响因子：
16.6
通讯作者：
Mann M
中科院分区：
文献类型：
--
作者：
Hansen FM;Tanzer MC;Brüning F;Bludau I;Stafford C;Schulman BA;Robles MS;Karayel O;Mann M
蛋白质泛素化几乎参与所有细胞过程。采用针对泛素衍生的二甘氨酸残基的抗体与质谱 (MS) 相结合的富集策略，使得对泛素信号传导的大规模研究成为可能。然而，到目前为止，数据独立采集 (DIA) 在单次运行分析灵敏度和数据完整性方面的作用尚未得到探索。在这里，我们开发了一个灵敏的工作流程，将基于二甘氨酸抗体的富集和优化的基于 Orbitrap 的 DIA 与包含超过 90,000 个二甘氨酸肽的综合光谱库相结合。该方法在蛋白酶体抑制剂处理的细胞的单次测量中识别出 35,000 个二甘氨酸肽——使数据依赖性采集的数量和定量准确性加倍。该工作流程应用于 TNF 信号传导，全面捕获已知位点，同时添加许多新位点。对昼夜节律周期中泛素化的深入、全系统研究发现了单个膜蛋白受体和转运蛋白内数百个循环泛素化位点和数十个循环泛素簇，强调了代谢和昼夜节律调节之间的新联系。蛋白质泛素化通常通过蛋白质组学进行研究，但数据独立采集 (DIA) 如何推进这些研究仍有待探索。在这里，作者表明 DIA 改善了泛素化位点的识别和量化，使他们能够表征人类细胞中的昼夜节律泛素组。
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. Protein ubiquitylation is often studied by proteomics but how data independent acquisition (DIA) may advance these studies remains to be explored. Here, the authors show that DIA improves ubiquitylation site identification and quantification, enabling them to characterize the circadian ubiquitinome in human cells.
登录
查看更多内容
影响因子：
16
作者：
Elia AE;Boardman AP;Wang DC;Huttlin EL;Everley RA;Dephoure N;Zhou C;Koren I;Gygi SP;Elledge SJ
通讯作者：
Elledge SJ
影响因子：
6
作者：
Collins PE;Mitxitorena I;Carmody RJ
通讯作者：
Carmody RJ
影响因子：
4
作者：
Doll, Sophia;Burlingame, Alma L.
通讯作者：
Burlingame, Alma L.
影响因子：
4.7
作者：
Babu, JR;Geetha, T;Wooten, MW
通讯作者：
Wooten, MW
影响因子：
56.9
作者：
Bruening, Franziska;Noya, Sara B.;Robles, Maria S.
通讯作者：
Robles, Maria S.