Isolation of protein subpopulations undergoing protein-protein interactions.
A new method is described for isolating and identifying proteins participating in protein-protein interactions in a complex mixture. The method uses a cyanogen bromide-activated Sepharose matrix to isolate proteins that are non-covalently bound to other proteins. Because the proteins are accessible to chemical manipulation, mass spectrometric identification of the proteins can yield information on specific classes of interacting proteins, such as calcium-dependent or substrate-dependent protein interactions. This permits selection of a subpopulation of proteins from a complex mixture on the basis of specified interaction criteria. The new method has the advantage of screening the entire proteome simultaneously, unlike the two-hybrid system or phage display, which can only detect proteins binding to a single bait protein at a time. The method was tested by selecting rat brain extract for proteins exhibiting calcium-dependent protein interactions. Of 12 proteins identified by mass spectrometry, eight were either known calcium-binding proteins or proteins with known calcium-dependent protein interactions, indicating that the method is capable of enriching a subpopulation of proteins from a complex mixture on the basis of a specific class of protein interactions. Because only naturally occurring interactions of proteins in their native state are observed, this method will have wide applicability to studies of protein interactions in tissue samples and autopsy specimens, for screening for perturbations of protein-protein interactions by signaling molecules, pharmacological agents or toxins, and screening for differences between cancerous and untransformed cells.
Nelson
Thomas J
TJ
Blanchette Rockefeller Neurosciences Institute, Rockville, Maryland 20850, USA. tjnelson@brni-jhu.org
Backlund
Peter S
PS
Yergey
Alfred L
AL
Alkon
Daniel L
DL
D000818:Animals; D001923:Brain Chemistry; D002846:Chromatography, Affinity; D002847:Chromatography, Agarose; D015180:Electrophoresis, Gel, Two-Dimensional; D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D020543:Proteome / Q000302:isolation & purification*; D051381:Rats
D046911:Macromolecular Substances; D009419:Nerve Tissue Proteins; D011506:Proteins; D020543:Proteome