A generic strategy to analyze the spatial organization of multi-protein complexes by cross-linking and mass spectrometry

Most cellular functions are performed by multi-protein complexes. The ident ity of the members of such complexes can now be determined by mass spectrom etry, Here we show that mass spectrometry can also be used in order to defi ne the spatial organization of these complexes. In this approach, component s of a protein complex are purified via molecular interactions using an aff inity tagged member and the purified complex is then partially cross-linked . The products are separated by gel electrophoresis and their constituent c omponents identified by mass spectrometry yielding nearest-neighbor relatio nships. In this study, a member of the yeast nuclear pore complex (Nup85p) was tagged and a six-member subcomplex of the pore was cross-linked and ana lyzed by 1D SDS-PAGE. Cross-linking reactions were optimized for yield and number of products. Analysis by MALDI mass spectrometry resulted in the ide ntification of protein constituents in the cross-linked bands even at a lev el of a few hundred femtomoles. Based on these results, a model of the spat ial organization of the complex was derived that was later supported by bio logical experiments, This work demonstrates, that the use of mass spectrome try is the method of choice for analyzing cross-linking experiments aiming on nearest neighbor relationships.