AbstractWe have developed quantitative cross-linking/mass spectrometry (QCLMS) to interrogate conformational rearrangements of proteins in solution. Our workflow was tested using a structurally well-described reference system, the human complement protein C3 and its activated cleavage product C3b. We found that small local conformational changes affect the yields of cross-linking residues that are near in space while larger conformational changes affect the detectability of cross-links. Distinguishing between minor and major changes required robust analysis based on replica analysis and a label-swapping procedure. By providing workflow, code of practice and a framework for semi-automated data processing, we lay the foundation for QCLMS as a tool to monitor the domain choreography that drives binary switching in many protein-protein interaction networks.AbbreviationsBS3Bis[sulfosuccinimidyl] suberateCLMSCross-linking/mass spectrometryFDRFalse discovery rateHCDHigher energy collision induced dissociationLC-MS/MSLiquid chromatography tandem mass spectrometryLTQLinear trap quadrupoleMS2Tandem mass spectrometryQCLMSQuantitative cross-linking/mass spectrometrySCXStrong cation exchange
Differential Tandem Mass Spectrometry-Based Cross-Linker: A New Approach for High Confidence in Identifying Protein Cross-Linking
