Proteomic Complex Detection Using Sedimentation

Protein−protein interactions are important in many cellular processes, but there are still relatively few methods to screen for novel protein complexes. Here we present a quantitative proteomics technique called ProCoDeS (Proteomic Complex Detection using Sedimentation) for profiling the sedimentation of a large number of proteins through a rate zonal centrifugation gradient. Proteins in a putative complex can be identified since they sediment faster than predicted from their monomer molecular weight. Using solubilized mitochondrial membrane proteins from Arabidopsis thaliana, the relative protein abundance in fractions of a rate zonal gradient was measured with the isotopic labeling reagent ICAT and electrospray mass spectrometry. Subunits of the same protein complex had very similar gradient distribution profiles, demonstrating the reproducibility of the quantitation method. The approximate size of the unknown complex can be inferred from its sedimentation rate relative to known protein complexes. ProCoDeS will be of use in screening extracts of tissues, cells, or organelle fractions to identify specific proteins in stable complexes that can be characterized by subsequent targeted techniques such as affinity tagging.