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“Zero‐length” cross‐linking in solid state as an approach for analysis of protein–protein interactions
Author(s) -
ElShafey Ahmed,
Tolic Nikola,
Young Malin M.,
Sale Kenneth,
Smith Richard D.,
Kery Vladimir
Publication year - 2006
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.051685706
Subject(s) - chemistry , dimer , peptide , protein structure , monomer , protein quaternary structure , trypsin , mass spectrometry , crystallography , amino acid , biochemistry , chromatography , enzyme , organic chemistry , protein subunit , gene , polymer
Abstract 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 H 2 16 O and H 2 18 O and then readily distinguished in mass spectra by characteristic 8 atomic mass unit (amu) shifts reflecting incorporation of two 18 O 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.