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| - A Generic Strategy To Analyze the SpatialOrganization of Multi-Protein Complexes byCross-Linking and Mass Spectrometry
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| - Most cellular functions are performed by multi-proteincomplexes. The identity of the members of such complexes can now be determined by mass spectrometry.Here we show that mass spectrometry can also be usedin order to define the spatial organization of these complexes. In this approach, components of a protein complexare purified via molecular interactions using an affinitytagged member and the purified complex is then partiallycross-linked. The products are separated by gel electrophoresis and their constituent components identified bymass spectrometry yielding nearest-neighbor relationships. In this study, a member of the yeast nuclear porecomplex (Nup85p) was tagged and a six-member subcomplex of the pore was cross-linked and analyzed by 1DSDS−PAGE. Cross-linking reactions were optimized foryield and number of products. Analysis by MALDI massspectrometry resulted in the identification of proteinconstituents in the cross-linked bands even at a level of afew hundred femtomoles. Based on these results, a modelof the spatial organization of the complex was derived thatwas later supported by biological experiments. This workdemonstrates, that the use of mass spectrometry is themethod of choice for analyzing cross-linking experimentsaiming on nearest neighbor relationships.
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