Increasing the Depth of Mass-Spectrometry-Based Structural Analysis of Protein Complexes through the Use of Multiple Cross-Linkers

Anal Chem. 2016 Apr 19;88(8):4461-9. doi: 10.1021/acs.analchem.6b00281. Epub 2016 Apr 4.

Abstract

Chemical cross-linking of proteins coupled with mass spectrometry (CXMS) is a powerful tool to study protein folding and to map the interfaces between interacting proteins. The most commonly used cross-linkers in CXMS are BS(3) and DSS, which have similar structures and generate the same linkages between pairs of lysine residues in spatial proximity. However, there are cases where no cross-linkable lysine pairs are present at certain regions of a protein or at the interface of two interacting proteins. In order to find the cross-linkers that can best complement the performance of BS(3) and DSS, we tested seven additional cross-linkers that either have different spacer arm structures or that target different amino acids (BS(2)G, EGS, AMAS, GMBS, Sulfo-GMBS, EDC, and TFCS). Using BSA, aldolase, the yeast H/ACA protein complex, and E. coli 70S ribosomes, we showed that, in terms of providing structural information not obtained through the use of BS(3) and DSS, EGS and Sulfo-GMBS worked better than the other cross-linkers that we tested. EGS generated a large number of cross-links not seen with the other amine-specific cross-linkers, possibly due to its hydrophilic spacer arm. We demonstrate that incorporating the cross-links contributed by the EGS and amine-sulfhydryl cross-linkers greatly increased the accuracy of Rosetta in docking the structure of the yeast H/ACA protein complex. Given the improved depth of useful information it can provide, we suggest that the multilinker CXMS approach should be used routinely when the amount of a sample permits.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cross-Linking Reagents / chemistry*
  • Mass Spectrometry / methods*
  • Models, Molecular
  • Protein Conformation
  • Protein Folding
  • Proteins / analysis*
  • Proteins / chemistry*

Substances

  • Cross-Linking Reagents
  • Proteins