PNNL

Abstract

Analyzing the architecture of protein complexes is a difficult task. Chemical cross-linking is often used in combination with mass spectrometric analysis to elucidate the interaction interfaces between proteins. We have developed a new approach for the analysis of interacting interfaces in protein complexes based on cross-linking in the solid state. Protein complexes are freeze-dried under vacuum and cross-links are introduced in the solid phase by dehydrating the protein in a non-water solvent, thus, creating peptide bonds between amino and carboxyl groups of the interacting peptides. Cross-linked proteins are digested into peptides with trypsin in both H216O and H218O and then readily distinguished in mass spectra by characteristic 8 atomic mass unit (amu) shifts reflecting incorporation of two 18O atoms into each C-terminus of proteolytic peptides. Computer analysis of mass spectrometry (MS) and MS/MS data is used to identify the cross-linked peptides.We demonstrated our method by cross-linking homooligomeric protein complexes alone or in a mixture of many other proteins. Cross-linking in the solid state was shown to be specific and reproducible. Glutathione-S-transferase (GST) from Schistosoma japonicum was studied in more detail. Twenty-seven unique intra-molecular and two inter-molecular cross-linked peptides were identified using tryptic mapping followed by LTQ-MS analysis. Identified cross-links were predominantly of amide origin, but six esters and thioesters were also found. Identified cross-linked peptides were validated by computational (visualization of cross-links in the three-dimensional [3D] structure of GST) and experimental (MS/MS) analyses. Most of the identified cross-links matched interacting peptides in the native 3D structure of GST indicating that the structure of GST and its oligomeric complex remained primarily intact after freeze drying. The pattern of oligomeric GST obtained in solid state was the same as that obtained in solution by Ru(II)Bpy32+ catalyzed, oxidative “zero-length” cross-linking, confirming that it is feasible to use our strategy for analyzing the molecular interfaces of interacting proteins or peptides.