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

2016 ◽  
Author(s):  
Zhuo A. Chen ◽  
Lutz Fischer ◽  
Salman Tahir ◽  
Jimi-Carlo Bukowski-Wills ◽  
Paul N. Barlow ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Conformational Changes ◽  
Cross Linking ◽  
Tandem Mass ◽  
Complement Protein ◽  
Protein Protein Interaction ◽  
Code Of Practice ◽  
Liquid Chromatography Tandem Mass ◽  
Binary Switching

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

scholarly journals Quantitative cross-linking/mass spectrometry reveals subtle protein conformational changes

2016 ◽  
Vol 1 ◽  
pp. 5 ◽  
Author(s):  
Zhuo Chen ◽  
Lutz Fischer ◽  
Salman Tahir ◽  
Jimi-Carlo Bukowski-Wills ◽  
Paul Barlow ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Conformational Changes ◽  
Complement C3 ◽  
Cross Linking ◽  
Complement Protein ◽  
Protein Protein Interaction ◽  
Code Of Practice ◽  
Final Maturation ◽  
Cross Links ◽  
Binary Switching

Quantitative cross-linking/mass spectrometry (QCLMS) probes protein structural dynamics in solution by quantitatively comparing the yields of cross-links between different conformational statuses. We have used QCLMS to understand the final maturation step of the proteasome lid and also to elucidate the structure of complement C3(H2O). Here we benchmark our workflow 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.

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