Abstract
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.
- 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