Abstract
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.
Highlights
A new method is described for isolating and identifying proteins participating in protein-protein interactions in a complex mixture
With the aid of high throughput robotics, the two-hybrid system has been adapted for proteomic screening
Initial attempts to identify protein-protein interactions using bifunctional cross-linking reagents resulted in complex mixtures of often insoluble aggregates containing multiple proteins, presumably caused by the small molecular size of the cross-linking reagent, which allowed it to bind to both interacting partners at multiple locations
Summary
Titration of CNBr-Sepharose—Cyanogen bromide activated Sepharose 4B (Amersham Biosciences) was rehydrated and washed three times with water before use. A quantity of rehydrated CNBr-Sepharose sufficient to bind 50% of the protein was added, and the sample was shaken at room temperature for 1 h. When the A280 of the eluate reached zero, the proteins retained by calcium-dependent interactions were eluted with 25 mM EGTA, desalted, and concentrated in a Centricon-3 ultrafiltration device, mixed 1:1 with IEF sample buffer (8.5 M urea, 2 M thiourea, 0.4% CHAPS, 0.5% immobilized pH gradient buffer (Amersham Biosciences) and 0.01% bromphenol blue), applied to an Immobiline pH 3–10 polyacrylamide isoelectric focusing strip that had been rehydrated with the same solution, and subjected to flatbed two-dimensional polyacrylamide gel electrophoresis (ExcelGel 12–14). The calmodulin-binding proteins were eluted with 25 mM EGTA, desalted, and concentrated in a Centricon-3 ultrafiltration device, separated by electrophoresis in a 4 –20% SDS-polyacrylamide gel, and blotted onto nitrocellulose membranes. Computer Analysis—Image quantitation, spot alignment, and molecular weight estimation were done using the image analysis program tnimage (entropy.brni-jhu.org)
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