After demonstrating the ability to identify SH2 domain-phosphoprotein interactions using recombinant proteins, a team led by researchers at the University of Houston set out to examine the feasibility of detecting in vivo protein-phosphoprotein interactions.
The researchers developed a cell based total protein assay to detect endogenous phosphoprotein-protein interactions mediated by tyrosine phosphorylation events in cancer cells. They hypothesize that the concentration of tyrosine phosphopeptides on the PepArray is much higher (1–4 µM) than the concentration achievable in vivo (in the range of pM or fM) which will enable the phosphopeptide to trap the respective protein complex involving the phosphor-motif.
To test the hypothesis they designed a tyrosine phosphopeptide array with 160 high affinity SH2 domain binding phosphor-motifs identified from a previous experiment involving recombinant SH2 domain screens. Each peptide probe was replicated 10 times on the chip with a control peptide for each replicate.
To detect and compare GRB2-phosphoprotein interactome networks, the phosphopeptide binding assay was performed on 4 different cell types derived from:
- Non-tumorigenic epithelium (MCF10A)
- ER positive tumor cells (MCF7)
- ER positive tumor cells (T47D)
- A breast metastatic (MDA-MB231) cell line
The experimental scheme of the cell lysate binding assay (a) and type of interactions expected from a complex mixture of total cellular proteins that might interact with each other through various phosphotyrosine binding domains and phosphotyrosine motifs (b) are shown in the figure below.
The image file obtained after scanning the chip (a), shown below, was measured by the absolute net signals of tyrosine phosphopeptide bound to GRB2 mediated protein complex detected by GRB2 protein specific primary antibody followed by the dye conjugated secondary antibody.
A rigorous data processing using in-house software programs with appropriate statistical analysis of data normalization was performed and high-ranked phosphopeptide probes interacting with endogenous GRB2 from cell lysates were selected (b).
A total of 57 phosphopeptide probes showed significant (2–3 fold) interaction differential between the cell types that was sufficient to distinguish one cell type from another.
Almost 70% of these interactions (40/57) were corroborated by previous studies and the rest are novel unreported interactions. This supports the researchers hypothesis of concentration mediated phosphopeptide-protein complex binding.
Based on Pepcyber database predictions, 24 interactions are directly GRB2 mediated, 17 interactions are either direct or in a complex with other proteins and 15 interactions are indirect with one or many interacting sandwich proteins as would be expected in the case of cell lysate with thousands of interacting proteins through different phosphotyrosine motifs.