Interactome and interface protocol (2IP): a novel strategy for high sensitivity topology mapping of protein complexes |
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Authors: | Weerasekera Rasanjala She Yi-Min Markham Kelly A Bai Yu Opalka Natacha Orlicky Stephen Sicheri Frank Kislinger Thomas Schmitt-Ulms Gerold |
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Institution: | Centre for Research in Neurodegenerative Diseases, University of Toronto, Ontario, Canada. |
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Abstract: | A few well-characterized protein assemblies aside, little is known about the topology and interfaces of multiconstituent protein complexes. Here we report on a novel indirect strategy for low-resolution topology mapping of protein complexes. Following crosslinking, purified protein complexes are subjected to chemical cleavage with cyanogen bromide (CNBr) and the resulting fragments are resolved by 2-D electrophoresis. The side-by-side comparison of a thus generated and a 2-D CNBr fragment map obtained from uncrosslinked material reveals candidate gel spots harboring crosslinked CNBr fragments. In-gel trypsinization and MALDI MS analysis of these informative spots identify the underlying crosslinked CNBr fragments based on unmodified tryptic peptides. Matching the cumulative theoretical molecular mass and predicted pI of these crosslinked CNBr fragments with original gel spot coordinates is required for confident crosslink assignment. The above strategy was successfully validated with the Escherichia coli RNA polymerase (RNAP) core complex and subsequently applied to query the quaternary structure of components of the yeast Skp1-Cdc53/Cullin-F box (SCF) ubiquitin ligase complex. This protocol requires low picomole sample quantities, can be applied to multisubunit protein complexes, and does not rely on specialized data mining software. |
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Keywords: | Mass spectra Protein structure characterization Quadrupole‐time of flight Two‐dimensional gel electrophoresis |
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