Proteomics Analysis Of DNA Damage Response

HPLC is an effective separation method, commonly used to increase and obtain proteome coverage in "bottom-up" shotgun proteomics. Despite many recent advances in phosphoproteome analysis, few studies have tried to compare the relative separation efficiency of different HPLC methods during phosphopeptide fractionation. To systematically evaluate different HPLC methods, enriched phosphopeptides with >90% purity were obtained from Hela cells by immobilized metal affinity chromatography (IMAC), and a fixed amount of phosphopeptides were subjected to various HPLC fractionation techniques before identification by RPLC-MS/MS analysis. The HPLC fractionation methods we used include strong cation exchange chromatography (SCX), hydrophilic interaction chromatography (HILIC) and electrostatic repulsion-hydrophilic interaction chromatography (ERLIC). We found that although between 4000-5000 unique phosphopeptides can be identified following any of the HPLC fraction method, each HPLC method yielded a considerable amount of non-overlapping unique phosphopeptides and had different separation characteristics as indicated by different selectivity, resolving power and orthogonality. Combining data from all the HPLC methods, we were able to identify 9069 unique phosphopeptides covering 9463 unique phosphorylation sites and 3260 unique phosphoproteins, indicating that different HPLC methods are complementary to each other, and can be used together in order to increase the phosphoproteome coverage. A number of new phosphorylation sites and novel phosphorylation motif information were also emerged from our study.The alkylating agent N-methyl-N-nitro-N-nitrosoguanidine (MNNG) is widely used in studying the mechanism of SN1 alkylating agent induced mutagenesis and carcinogenesis. MNNG can induce cellular DNA damage response (DDR) which is a very complex process, and many biological pathways, such as DNA repair, cell cycle arrest, chromatin aberration and apoptosis, are involved. It is well known that MutSa, a mismatch repair protein consist of MSH2 and MSH6 subunits, can recognize alkylated bases 6MeG/T caused by MNNG and play key roles in the initiating steps of DNA damage signaling. To investigate the molecular mechanism of DDR and explore the involvement of mismatch proteins in apoptosis, we employed high-throughput proteomic techniques, including of SILAC-based quantitative mass spectrometry, nuclear proteins fractionation, phosphopeptide enrichment and multidimensional chromatography, to detect changes in phosphorylation stoichiometry and protein abundance following MNNG treatment of TK6 (proficient in MMR proteins, sensitive to MNNG-induced apoptosis) and MT1 (contains a hMSH6 mutation, resistant to MNNG-induced apoptosis) cells. MNNG-induced changes in 1108 phosphorylation-sites and 794 proteins were obtained from TK6 cells, and 901 phosphorylation-sites and 724 proteins were obtained from MT1 cells. The comprehensive quantitative proteomics data provided new information about the complicated molecular mechanisms and biological pathways of MNNG-induced cellular responses.