Novo Nordisk Foundation Center for Protein Research, Proteomics Program, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3b, 2200, Copenhagen, Denmark.
Biognosys AG, Wagistrasse 21, 8952, Schlieren, Switzerland.
Nat Commun. 2020 Feb 7;11(1):787. doi: 10.1038/s41467-020-14609-1.
Quantitative phosphoproteomics has transformed investigations of cell signaling, but it remains challenging to scale the technology for high-throughput analyses. Here we report a rapid and reproducible approach to analyze hundreds of phosphoproteomes using data-independent acquisition (DIA) with an accurate site localization score incorporated into Spectronaut. DIA-based phosphoproteomics achieves an order of magnitude broader dynamic range, higher reproducibility of identification, and improved sensitivity and accuracy of quantification compared to state-of-the-art data-dependent acquisition (DDA)-based phosphoproteomics. Notably, direct DIA without the need of spectral libraries performs close to analyses using project-specific libraries, quantifying > 20,000 phosphopeptides in 15 min single-shot LC-MS analysis per condition. Adaptation of a 3D multiple regression model-based algorithm enables global determination of phosphorylation site stoichiometry in DIA. Scalability of the DIA approach is demonstrated by systematically analyzing the effects of thirty kinase inhibitors in context of epidermal growth factor (EGF) signaling showing that specific protein kinases mediate EGF-dependent phospho-regulation.
定量磷酸化蛋白质组学已经改变了细胞信号转导的研究，但该技术仍难以扩展到高通量分析。在这里，我们报告了一种快速且可重复的方法，使用包含在 Spectronaut 中的准确位点定位分数的无依赖性数据采集（DIA）来分析数百个磷酸化蛋白质组。与最先进的基于数据依赖性采集（DDA）的磷酸化蛋白质组学相比，基于 DIA 的磷酸化蛋白质组学具有更广泛的动态范围、更高的鉴定重复性、以及更好的灵敏度和定量准确性。值得注意的是，直接进行 DIA 而无需使用光谱库，其性能与使用特定项目的库进行分析非常接近，在 15 分钟的单 Shot LC-MS 分析中，每个条件下可以定量超过 20000 个磷酸肽。基于 3D 多元回归模型算法的适应性允许在 DIA 中全局确定磷酸化位点的化学计量。通过系统地分析三十种激酶抑制剂在表皮生长因子（EGF）信号转导中的作用，证明了 DIA 方法的可扩展性，表明特定的蛋白激酶介导了 EGF 依赖性磷酸化调节。
