Abstract 2740: Identification of new binding partners of the DNA repair protein MGMT using a proteomic discovery-based approach in glioblastoma

Background: Glioblastoma multiforme (GBM) is characterized by aberrant angiogenesis and widespread invasion through the brain parenchyma. The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) has been extensively characterized for its role in resistance to alkylating agents used in treatment of GBM. Our team discovered an inverse relationship between expression of MGMT and GBM angiogenesis and invasion. To gain new insights into how MGMT affects angiogenesis and invasion, we used a proteomic-based approach integrated with bioinformatics analysis to identify potential MGMT-binding partners (BPs) for the first time in GBM. Methods: We used T98G, a human GBM cell line with constitutive expression of MGMT and performed direct immunoprecipitation (IP) of endogenous MGMT using an anti-MGMT antibody or the isotype control. Following elution of the antibody, proteins were resolved by SDS-PAGE, stained, excised from the gel then subjected to trypsin digestion and identified by liquid chromatography-tandem mass spectrometry using the LTQ-Orbitrap Elite mass analyzer. The resulting tryptic peptides were purified and identified by LC-tandem mass spectrometry (MS/MS). The resultant MS/MS spectra were searched against a proteome database for peptide matching and protein identification. Proteins identified with high confidence (Scaffold software) were used to construct the biological network of MGMT-BPs in GBM using the Build Network tool provided by MetaCore. Results: We identified a total of 186 MGMT-BPs, which were not identified in the elution from the isotype control. Using gene ontology (GO) database, we determined the function and biological role of identified proteins (mitochondrial metabolism, DNA repair and replication, ubiquitin pathway, transcription regulators, RNA post-transcriptional processing, transcriptional splicing, protein biosynthesis and trafficking, cellular metabolism, cell cycle and division, response to stress and cell death, cell migration and invasion). The list of new BPs was uploaded to MetaCore and the most relevant biological process was enriched based on public GO databases. Among the top proteins identified with a very high confidence, we found newly identified MGMT-BPs, which may underlie the role of MGMT in angiogenesis and invasion, namely the splicing factor heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), known for its role in the packaging of pre-mRNA into hnRNP particles and alternative splicing of angiogenic