Cookies on this website

We use cookies to ensure that we give you the best experience on our website. If you click 'Accept all cookies' we'll assume that you are happy to receive all cookies and you won't see this message again. If you click 'Reject all non-essential cookies' only necessary cookies providing core functionality such as security, network management, and accessibility will be enabled. Click 'Find out more' for information on how to change your cookie settings.

Recent developments in purification strategies, together with mass spectrometry (MS)-based proteomics, have identified numerous in vivo protein complexes and suggest the existence of many others. Standard proteomics techniques are, however, unable to describe the overall stoichiometry, subunit interactions and organization of these assemblies, because many are heterogeneous, are present at relatively low cellular abundance and are frequently difficult to isolate. We combine two existing methodologies to tackle these challenges: tandem affinity purification to isolate sufficient quantities of highly pure native complexes, and MS of the intact assemblies and subcomplexes to determine their structural organization. We optimized our protocol with two protein assemblies from Saccharomyces cerevisiae (scavenger decapping and nuclear cap-binding complexes), establishing subunit stoichiometry and identifying substoichiometric binding. We then targeted the yeast exosome, a nuclease with ten different subunits, and found that by generating subcomplexes, a three-dimensional interaction map could be derived, demonstrating the utility of our approach for large, heterogeneous cellular complexes.

Original publication

DOI

10.1038/sj.embor.7400702

Type

Journal article

Journal

EMBO Rep

Publication Date

06/2006

Volume

7

Pages

605 - 610

Keywords

Chromatography, Affinity, Dimerization, Exoribonucleases, Fungal Proteins, Gene Deletion, Genes, Fungal, Isotope Labeling, Mass Spectrometry, Models, Biological, Multiprotein Complexes, Protein Binding, Protein Interaction Mapping, Protein Structure, Quaternary, Protein Subunits, Proteome, Proteomics, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins