Dörig Christian, Marulli Cathy, Peskett Thomas, Volkmar Norbert, Pantolini Lorenzo, Studer Gabriel, Paleari Camilla, Frommelt Fabian, Schwede Torsten, de Souza Natalie, Barral Yves, Picotti Paola
Institute of Molecular Systems Biology, Department of Biology, ETH Zurich, Zurich, Switzerland.
Institute of Biochemistry, Department of Biology, ETH Zurich, Zurich, Switzerland.
Nat Biotechnol. 2024 Oct 16. doi: 10.1038/s41587-024-02432-8.
Methods to systematically monitor protein complex dynamics are needed. We introduce serial ultrafiltration combined with limited proteolysis-coupled mass spectrometry (FLiP-MS), a structural proteomics workflow that generates a library of peptide markers specific to changes in PPIs by probing differences in protease susceptibility between complex-bound and monomeric forms of proteins. The library includes markers mapping to protein-binding interfaces and markers reporting on structural changes that accompany PPI changes. Integrating the marker library with LiP-MS data allows for global profiling of protein-protein interactions (PPIs) from unfractionated lysates. We apply FLiP-MS to Saccharomyces cerevisiae and probe changes in protein complex dynamics after DNA replication stress, identifying links between Spt-Ada-Gcn5 acetyltransferase activity and the assembly state of several complexes. FLiP-MS enables protein complex dynamics to be probed on any perturbation, proteome-wide, at high throughput, with peptide-level structural resolution and informing on occupancy of binding interfaces, thus providing both global and molecular views of a system under study.
需要有系统地监测蛋白质复合物动态变化的方法。我们引入了串联超滤结合有限蛋白水解偶联质谱法（FLiP-MS），这是一种结构蛋白质组学工作流程，通过探测蛋白质复合物结合形式和单体形式之间蛋白酶敏感性的差异，生成特定于蛋白质-蛋白质相互作用（PPI）变化的肽段标记库。该库包括映射到蛋白质结合界面的标记以及报告伴随PPI变化的结构变化的标记。将标记库与有限蛋白水解质谱法（LiP-MS）数据相结合，能够对未分级裂解物中的蛋白质-蛋白质相互作用进行全局分析。我们将FLiP-MS应用于酿酒酵母，并探测DNA复制应激后蛋白质复合物动态变化，确定了Spt-Ada-Gcn5乙酰转移酶活性与几种复合物组装状态之间的联系。FLiP-MS能够在蛋白质组范围内、高通量地、以肽段水平的结构分辨率探测任何扰动下的蛋白质复合物动态变化，并提供结合界面占有率信息，从而提供所研究系统的全局和分子视图。
