Proteomics Approach for Identifying Interacting Partners of a C-Terminal Functional Peptide Derived from the Tumor Suppressor, p21cip/waf1
With the recent technological innovations of proteomics, protein separation followed by mass spectrometry (MS) has become the technique of choice in identifying and validating potential drug targets. As a part of our drug discovery program, we tested the validity of this approach by conducting a simple affinity extraction followed by mass spectrometric (MS) analysis of the components isolated. For this purpose, we chose the C-terminal peptide of the tumor suppressor p21cip/waf1, which is known to bind proliferating cell nuclear antigen (PCNA) and cause cell cycle arrest . Using Jurkat-E cell lysate, affinity extraction of PCNA and its binding partners was carried out by spiking streptavidin agarose beads pre-conjugated with biotinylated p21-derived peptide(s). Using tryptic digests of entire affinity extracts and differential micro-capillary LC/MS/MS, or difference 2D gels combined with in-gel tryptic digests and MALDI-TOF MS, we have identified binding partners of the p21 C-terminal peptide, or of its complex with PCNA. Results from the above experiments were confirmed either by reciprocal affinity extraction and/or Western blotting with respective antibodies. This study suggests that peptides obtained from intracellular functional screens could also serve as efficient baits to affinity extract target proteins and map mammalian cell protein interaction networks.
KeywordsAgarose Acetonitrile Biotin Biot
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