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 identity of the members of such complexes can now be determined by mass spectrometry. Here we show that mass spectrometry can also be used in order to define the spatial organization of these complexes. In this approach, components of a protein complex are purified via molecular interactions using an affinity tagged member and the purified complex is then partially cross-linked. The products are separated by gel electrophoresis and their constituent components identified by mass spectrometry yielding nearest-neighbor relationships. In this study, a member of the yeast nuclear pore complex (Nup85p) was tagged and a six-member sub-complex of the pore was cross-linked and analyzed by 1D SDS-PAGE. Cross-linking reactions were optimized for yield and number of products. Analysis by MALDI mass spectrometry resulted in the identification of protein constituents in the cross-linked bands even at a level of a few hundred femtomoles. Based on these results, a model of the spatial organization of the complex was derived that was later supported by biological experiments. This work demonstrates, that the use of mass spectrometry is the method of choice for analyzing cross-linking experiments aiming on nearest neighbor relationships.
Rappsilber
J
J
Protein Interaction Laboratory, University of Southern Denmark, Odense, Denmark.
Siniossoglou
S
S
Hurt
E C
EC
Mann
M
M
D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000737:chemistry*; D010449:Peptide Mapping; D018272:Porins / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry* / Q000187:drug effects; D029701:Saccharomyces cerevisiae Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D015003:Yeasts / Q000737:chemistry*
D003432:Cross-Linking Reagents; C106899:NUP85 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D018272:Porins; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins