Selective enrichment and identification of cross-linked peptides to study 3-D structures of protein complexes by mass spectrometry.
J Proteomics
; 75(7): 2205-15, 2012 Apr 03.
Article
em En
| MEDLINE
| ID: mdl-22326961
ABSTRACT
Chemical cross-linking of protein complexes combined with mass spectrometry is a powerful approach to obtain 3-D structural information by revealing amino residues that are in close spatial proximity. To increase the efficiency of mass spectrometric analysis, we have demonstrated the selective enrichment of cross-linked peptides from the 350 kDa protein complex RNA polymerase (RNAP) from Bacillus subtilis. Bis(succinimidyl)-3-azidomethyl glutarate was used as a cross-linker along with an azide-reactive cyclooctyne-conjugated resin to capture target peptides. Subsequently released peptides were fractionated by strong cation exchange chromatography and subjected to LC-MS/MS. We mapped 10 different intersubunit and 24 intrasubunit cross-links by xComb database searching supplied with stringent criteria for confirmation of the proposed structure of candidate cross-linked peptides. The cross-links fit into a homology model of RNAP. Cross-links between ß lobe 1 and the ß' downstream jaw, and cross-links involving the N-terminal and C-terminal parts of the α subunits suggest conformational flexibility. The analytical strategy presented here can be applied to map protein-protein interactions at the amino acid level in biological assemblies of similar complexity. Our approach enables the exploration of alternative peptide fragmentation techniques that may further facilitate cross-link analysis.
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Base de dados:
MEDLINE
Assunto principal:
Peptídeos
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Bacillus subtilis
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Proteínas de Bactérias
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RNA Polimerases Dirigidas por DNA
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Modelos Moleculares
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Bases de Dados de Proteínas
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Homologia Estrutural de Proteína
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article