Isolation of Protein Subpopulations Undergoing Protein-Protein Interactions

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 bromideactivated 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.

Hippocampal protein‐protein interactions in spatial memory

“…In a previous report (Nelson et al, 2002), we showed that by eluting the bound proteins with EGTA instead of urea, the selection method can be used to screen for calcium-dependent protein interactions. Of 12 proteins identified in this earlier study, 8 were either known calcium-binding proteins or proteins with known calcium-dependent protein interactions, indicating that the method was capable of enriching a subpopulation of proteins from a complex mixture on the basis of specific interaction criteria.…”

“…Even methods used for large-scale functional genomics, such as the yeast two-hybrid system (Fields and Song, 1989;Schwinkowski and Fields, 2000;Uetz et al, 2000) and phage display (Smith, 1985;Rossenu et al, 1997), cannot be adapted to search for protein interactions that occur as a physiological response in specialized tissues because of their reliance on single-celled organisms and cloned proteins. Therefore, we used a novel separation method (Nelson et al, 2002) to isolate a subpopulation of proteins that participates in protein-protein interactions. This permitted the identification of 16 proteins exhibiting heretofore unknown memory-specific changes in protein interactions.…”

Warburg Effect’s Manifestation in Aggressive Pheochromocytomas and Paragangliomas: Insights from a Mouse Cell Model Applied to Human Tumor Tissue

“…For those, LC/MS/MS analysis was performed as described previously [65]. The resulting tandem mass spectra were extracted using BioWorks v3, and the MS/MS spectra were analyzed by both Mascot (version 2.3; Matrix Science, London, UK) [64] and X!…”

Proteomics in 2002: A Year of Technical Development and Wide-Ranging Applications

“…The spots observed to be different between the normal versus the mutant pull-downs were identified by MS. Fifteen proteins that interacted with the C-terminal tail of the receptor were observed. A method, based on cyanogens bromide-activated Sepharose matrix, was developed by Nelson et al (191) for isolating interacting proteins. In this method, activated Sepharose is added to bind 50% of the proteins present in a mixture.…”