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.

需要系统监测蛋白质复合物动力学的方法。我们介绍了连续超滤结合有限蛋白水解偶联质谱 （FLiP-MS），这是一种结构蛋白质组学工作流程，通过探测复合物结合和单体蛋白质之间的蛋白酶易感性差异，生成对 PPI 变化具有特异性的肽标志物库。该文库包括映射到蛋白质结合界面的标记物和报告伴随 PPI 变化的结构变化的标记物。将标记物库与 LiP-MS 数据整合，可以对未分级裂解物中的蛋白质-蛋白质相互作用 （PPI） 进行整体分析。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力学的变化，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 能够在任何扰动下以高通量、蛋白质组范围、肽水平的结构分辨率探测蛋白质复合物动力学，并告知结合界面的占用情况，从而提供所研究系统的全局和分子视图。