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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
3
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pubmed:dateCreated |
2006-2-27
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pubmed:abstractText |
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.
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pubmed:commentsCorrections |
http://linkedlifedata.com/resource/pubmed/commentcorrection/16501223-10339534,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16501223-10811876,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16501223-10975572,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16501223-12021454,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16501223-12139305,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16501223-12236350,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16501223-12716128,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16501223-12888339,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16501223-12892908,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16501223-14696200,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16501223-14706816,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16501223-15253422,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16501223-15372733,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16501223-15530987,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16501223-15623310,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16501223-15649034,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16501223-2344038,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16501223-4436300,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16501223-7538846,
http://linkedlifedata.com/resource/pubmed/commentcorrection/16501223-8744570
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:month |
Mar
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pubmed:issn |
0961-8368
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pubmed:author |
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pubmed:issnType |
Print
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pubmed:volume |
15
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
429-40
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pubmed:dateRevised |
2009-11-18
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pubmed:meshHeading |
pubmed-meshheading:16501223-Acetone,
pubmed-meshheading:16501223-Binding Sites,
pubmed-meshheading:16501223-Carbodiimides,
pubmed-meshheading:16501223-Cross-Linking Reagents,
pubmed-meshheading:16501223-Freeze Drying,
pubmed-meshheading:16501223-Glutathione Transferase,
pubmed-meshheading:16501223-Mass Spectrometry,
pubmed-meshheading:16501223-Models, Molecular,
pubmed-meshheading:16501223-Multiprotein Complexes,
pubmed-meshheading:16501223-Oxygen Isotopes,
pubmed-meshheading:16501223-Peptides,
pubmed-meshheading:16501223-Protein Interaction Mapping
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pubmed:year |
2006
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pubmed:articleTitle |
"Zero-length" cross-linking in solid state as an approach for analysis of protein-protein interactions.
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pubmed:affiliation |
Cell Biology and Biochemistry, Pacific Northwest National Laboratory, 902 Battelle Blvd., P.O. Box 999, K4-12, Richland, WA 99354, USA.
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pubmed:publicationType |
Journal Article,
Evaluation Studies
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