biostudies-literatureDorig CEuropean Research CouncilPromedica StiftungEC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)1562-1576https://www.ebi.ac.uk/biostudies/studies/S-EPMC12440823biostudies-literatureUnknown43(9)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, identifyNature biotechnologyGlobal profiling of protein complex dynamics with an experimental library of protein interaction markers.PMC124408231508/M866004823839Schwede TDorig CVolkmar Nde Souza NBarral YStuder GPantolini LPaleari CFrommelt FMarulli CPicotti PPeskett TfalseGlobal profiling of protein complex dynamics with an experimental library of protein interaction markers.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, identify2025-01-01T00:00:00Z2025 Sep2026-06-03T07:59:58.094Z2026-04-26T03:12:02.137ZS-EPMC124408233941505910.1038/s41587-024-02432-8