posted on 2007-03-01, 00:00authored byNicholas T. Hartman, Francesca Sicilia, Kathryn S. Lilley, Paul Dupree
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.