Quantitative Cross-Linking/Mass Spectrometry Reveals Subtle Protein Conformational Changes

We 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. Abbreviations BS3 Bis[sulfosuccinimidyl] suberate CLMS Cross-linking/mass spectrometry FDR False discovery rate HCD Higher energy collision induced dissociation LC-MS/MS Liquid chromatography tandem mass spectrometry LTQ Linear trap quadrupole MS2 Tandem mass spectrometry QCLMS Quantitative cross-linking/mass spectrometry SCX Strong cation exchange

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