UC Riverside

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful platform for the identification and characterizations of chemical modifications on DNA, RNA and proteins. With the rapid development of epigenetics and epitranscriptomics fields, a large number of novel DNA, RNA, and histone modifications are discovered. There is a high demand for sensitive and accurate quantifications of epigenetic, epitranscriptomic modifications and their modulators to better understand their biological roles in multiple cellular processes. The focus of this dissertation is on LC-MS/MS-based quantitative analysis of glutathione-conjugated abasic sites and epigenetic/epitranscriptomic reader, writer and eraser (RWE) proteins.In Chapter 2, I identified the presence of appreciable levels of 3-glutathionyl-2,3-dideoxyribose (GS-ddR), which is a novel endogenous DNA lesion, in cellular DNA and developed an LC-MS/MS method to quantify GS-ddR in genomic DNA of cultured human cells. The quantification results showed the induction of GS-ddR by an alkylating agent, N-methyl-N-nitrosourea (MNU) in mammalian cells. In Chapter 3, by employing a parallel-reaction monitoring (PRM)-based targeted proteomics method, I performed a comprehensive quantitative assessment of 154 epitranscriptomic RWE proteins during the time course of osteogenic differentiation of H9 human embryonic stem cells (ESCs). The work demonstrated the potential roles of epitranscriptomic RWE protein in osteogenesis and offered novel insight into the mechanisms of osteogenesis. In Chapter 4, I investigated how H3K36me3 modulates the chromatin occupancies of epitranscriptomic RWE proteins. The work revealed cross-talks between H3K36me3 and epitranscriptomic RWE proteins and uncovered potential roles of these RWE proteins in H3K36me3-mediated biological processes. In Chapter 5, I established a high-throughput LC-PRM method for targeted profiling of 151 histone methylation and acetylation RWE proteins and investigated how H3K36me3 and m6A modulates the chromatin occupancies of histone methylation and acetylation RWE proteins. Together, the research described in this dissertation documented the power of LC-MS/MS-based quantitative analysis in the identification and characterizations of epigenetic/epitranscriptomic modifications and their modulators.