"Zero-length" cross-linking in solid state as an approach for analysis of protein-protein interactions.
We have developed a new approach for the analysis of interacting interfaces in protein complexes and protein quaternary structure 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 nonaqueous solvent creating peptide bonds between amino and carboxyl groups of the interacting peptides. Cross-linked proteins are digested into peptides with trypsin in both H2(16)O and H(2)18O 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 specificity and reproducibility of our method by cross-linking homo-oligomeric protein complexes of glutathione-S-transferase (GST) from Schistosoma japonicum alone or in a mixture of many other proteins. Identified cross-links were predominantly of amide origin, but six esters and thioesters were also found. The cross-linked peptides were validated against the GST monomer and dimer X-ray structures and by experimental (MS/MS) analyses. Some 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) Bpy(3)2+ 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.
El-Shafey
Ahmed
A
Cell Biology and Biochemistry, Pacific Northwest National Laboratory, 902 Battelle Blvd., P.O. Box 999, K4-12, Richland, WA 99354, USA.
Tolic
Nikola
N
Young
Malin M
MM
Sale
Kenneth
K
Smith
Richard D
RD
Kery
Vladimir
V
D000096:Acetone / Q000737:chemistry; D001665:Binding Sites; D002234:Carbodiimides / Q000737:chemistry; D003432:Cross-Linking Reagents; D005612:Freeze Drying; D005982:Glutathione Transferase / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry*; D010103:Oxygen Isotopes; D010455:Peptides / Q000032:analysis; D025941:Protein Interaction Mapping / Q000379:methods*
D002234:Carbodiimides; D003432:Cross-Linking Reagents; D046912:Multiprotein Complexes; D010103:Oxygen Isotopes; D010455:Peptides; D000096:Acetone; D005982:Glutathione Transferase