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"Zero-length" cross-linking in solid state as an approach for analysis of protein–protein interactions

¹ Pacific Northwest National Laboratory (PNNL), Richland, Washington 99354, USA
² Sandia National Laboratory, Livermore, California 94550, USA

(RECEIVED July 6, 2005; FINAL REVISION December 8, 2005; ACCEPTED December 9, 2005)

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 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 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) 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.

Keywords: cross-linking; mass spectrometry; protein complex; protein folding; protein–protein interactions; protein structure; solid state; ¹⁸O labeling

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051685706.

Reprint requests to: Vladimir Kery, Cell Biology and Biochemistry, Pacific Northwest National Laboratory, 902 Battelle Blvd., P.O. Box 999, K4-12, Richland, WA 99354, USA; e-mail: Vladimir.Kery{at}pnl.gov; fax: (509) 372-6455.

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