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Zoekopdracht: faculteit: "FNWI" en publicatiejaar: "2012"

AuteursH. Buncherd, M.A. Nessen, N. Nouse, S.K. Stelder, W. Roseboom, H.L. Dekker, J.C. Arents, L.E. Smeenk, M.J. Wanner, J.H. van Maarseveen, X. Yang, P.J. Lewis, L.J. de Koning, C.G. de Koster, L. de Jong
TitelSelective enrichment and identification of cross-linked peptides to study 3-D structures of protein complexes by mass spectrometry.
TijdschriftJ Proteomics
FaculteitFaculteit der Natuurwetenschappen, Wiskunde en Informatica
Instituut/afd.FNWI: Van 't Hoff Institute for Molecular Sciences (HIMS)
FNWI: Swammerdam Institute for Life Sciences (SILS)
SamenvattingChemical 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 350kDa 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 beta lobe 1 and the beta' downstream jaw, and cross-links involving the N-terminal and C-terminal parts of the alpha 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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