Arsenite exposure in drinking water and food has long been a health concern for decades because of its cellular toxicity and carcinogenic effects in inducing various cancers and cardiovascular diseases. This research focuses on exploring underlying mechanism accounting for arsenite toxicity, in the aspect of targeting E3 ubiquitin ligases to suppress ubiquitination of substrate proteins, which serve as important signals in multiple functional pathways and proteasome-mediated degradation of proteins.

In chapter 1, I briefly described some background of arsenite toxicity and the role of ubiquitination in cells. I also introduced the application of LC-MS for the analysis of proteome, typically ubiquitin-modified proteome in this study. By employing LC-MS/MS couple with SILAC labeling and affinity enrichment, I was able to identify ~1500 ubiquitinated peptides, of which ~40% peptides were decreased upon arsenite exposure. I did a KEGG pathway analysis and functional categories of the identified proteins and was able to find some interested protein targets that were studied in chapter 2 and chapter 3, including FANCD2 and HMGCR, respectively.

The LC-MS/MS results showed a 50% decrease of FANCD2-K561 ubiquitination upon arsenite treatment. I further demonstrated, for the first time, that arsenite targets the RING finger domain of the E3 ubiquitin ligase, FANCL, to suppress FANCD2 ubiquitination, using MALDI-TOF mass spectrometry and biotin-As pull down coupled with Western blot. This interaction was also found to impair chromatin localization of FANCD2, which is crucial for recruiting downstream repair factors for DNA interstrand crosslink repair. Therefore, for the first time, I revealed a role of arsenite in perturbing the Fanconi anemia pathway for DNA interstrand crosslink repair by targeting the RING finger of FANCL E3 ubiquitin ligase and inhibit FANCD2 monoubiquitination.

The LC-MS/MS results also detected a 70% decrease of HMGCR-K248 upon arsenite treatment, which is important for HMGCR degradation and maintain cholesterol homeostasis in mammalian cells. Indeed I found that arsenite could stabilize HMGCR protein level by inhibiting its ubiquitination. This finding suggests a role of arsenite in affecting the cholesterol biosynthesis pathway.

In chapter 4, I further expanded the role of arsenite in targeting E3 ubiquitin ligase by measuring the interaction between arsenite and the E3 ubiquitin ligase RBX1, which is responsible for Nrf2 ubiquitination. This provides a novel insight in arsenite-mediated activation of antioxidant response.

The advances in mass spectrometry (MS) instrumentation have rendered MS an indispensable tool for structure elucidation, quantification, and assessment of the biological consequences of DNA modifications. DNA encodes genetic information that is used in the development and functioning of essentially all known organisms. Changes in DNA could give rise to compromised transmission of genetic information. Abnormal modifications are generally harmful to cells and will be converted to permanent genetic damage if they evade DNA repair, whereas epigenetic DNA modifications are regulators of cell function that influence chromatin structure and control the levels of gene expression. The emphasis of this dissertation is on the application of MS, together with biological techniques, for assessing the occurrence and biological consequences of these two major types of DNA modifications.

After an introduction presented in Chapter 1, Chapter 2 focuses on monitoring the enzymatic removal of 8-methoxypsoralen-induced DNA interstrand cross-links (ICLs) in cultured mammalian cells by using an HPLC coupled with tandem-MS approach. With genetic depletion of DNA repair proteins, we also pinpointed the role of nucleotide excision repair pathway in removing DNA ICLs. Chapters 3 and 4 discuss the development of an HPLC-MS/MS/MS method, along with the isotope dilution technique, for accurate measurements of 5-hydroxymethyl, 5-formyl and 5-carboxyl derivatives of 2'-deoxycytidine and 5-hydroxymethyl-2'-deoxyuridine (5-hmdU) in genomic DNA of cultured human cells, mammalian tissues and plant tissues. This is the first direct quantitative comparison between the levels of multiple 5-mdC derivatives in mammalian tissues and the first report of their presence in the model plant Arabidopsis. Chapter 5 describes the combination of this method with other bioanalytical techniques for studying the mechanisms of arsenic epigenotoxicity. We found that arsenite could bind directly to the zinc fingers of Tet proteins and substantially impair the catalytic efficiency of Tet proteins in oxidizing 5-mC both in vitro and in cells. In Chapter 6, we report the application of LC-MS/MS for quantifying β-D-glucosyl-5-hydroxymethyl- 2'-deoxyuridine (dJ) in Trypanosoma brucei. The results provide evidence that dJ binding proteins play an important role in oxidizing thymidine to form 5-hmdU, which then facilitates the generation of dJ.

Mass spectrometry (MS)-based bottom-up proteomics and quantitative proteomic labeling strategies have led to unprecedented insights into systems biology and provided invaluable resources as a multifaceted analytical tool. We have utilized such techniques to analyze small GTPases of the Ras superfamily, which represent a class of crucial signaling molecules in cells, and the aberrant regulation of their expressions is implicated with various types of human diseases. In this dissertation, I report the development and applications of novel targeted quantitative proteomic methods for high-throughput and reproducible profiling of small GTPases in cultured human cells and patient-derived brain tissues that carry disease-related changes.

In Chapter 2, I describe the development of a novel scheduled multiple-reaction monitoring (MRM)-based targeted quantitative proteomic method, in conjunction with stable isotope labeling by amino acids in cell culture (SILAC) for the quantification of more than 90 small GTPases in the paired primary/metastatic melanoma cell lines. The data reveal previously unrecognized roles of RAB38 in promoting melanoma metastasis in vitro.

In Chapter 3, the established scheduled MRM-based method was further applied to assess the differential expression of small GTPases in wild-type MCF-7 and the paired tamoxifen-resistant breast cancer cells. The method facilitated robust quantification of 96 small GTPases, among which down-regulation of RAB31 was analyzed further and demonstrated to play a role in the development of acquired tamoxifen resistance.

In Chapter 4, we extended the use of the scheduled MRM method to comprehensively investigate the differential expression of small GTPases in paired primary/metastatic colorectal cancer cell (CRC) lines SW480 and SW620. With this approach, 83 small GTPases were robustly quantified, leading to the identification of SAR1B as a potential suppressor for CRC metastasis. We also showed that diminished SAR1B expression could stimulate epithelial–mesenchymal transition (EMT), thereby promoting motility and in vitro metastasis of SW480 cells.

In Chapter 5, I describe the development of a novel targeted quantitative proteomic assay based on MRM and the use of crude synthetic stable isotope-labeled (SIL) peptides as internal standards (IS) and surrogate standards (SS). By using this approach, we quantified ~80 small GTPases from lysates of frontal cortex from post-mortem Alzheimer’s disease (AD) patient brain tissue samples. The method displayed excellent throughput, sensitivity and reproducibility. Furthermore, we observed that the protein expression levels of Rab3A/C, Rab4A/B and Rab27B proteins, which are involved with synaptic and secretory vesicles, increased with degree of disease severity. The MRM quantification results were further verified by Western blotting.

Guanine quadruplex (G4) structures are nonconical DNA conformations that play crucial roles in various cellular processes, including DNA replication and transcription regulation. G4 structures are frequently found in promoter regions of oncogenes, which render G4 structures promising therapeutic targets. However, the mechanisms through which G4 structures regulate biological functions remain underexplored. This dissertation focuses on the development of novel quantitative proteomic methods to identify putative G4-binding proteins, to characterize novel G4-binding proteins, to explore cellular modifiers of G4 stability, and to discover of novel biomarkers in breast cancer cells. In chapter 2, we employed an affinity-based quantitative proteomics analysis to identify novel G4-binding proteins. By utilizing three different biotinylated G4-forming oligonucleotides as probes, I achieved a comprehensive analysis of proteins interacting with various G4 conformations, which led to the discovery of over 30 G4-binding proteins. Building upon the pull-down experiments, I introduced, in Chapter 3, the use of photo-crosslinking G4 probes to capture G4-binding proteins. This approach enabled the capture of weak and transient interactions, where harsh washing conditions eliminate protein-protein interactions during the pull-down process. By Using this method, I identified 99 putative G4-binding proteins, and I also characterized one of these proteins, HELLS as a novel G4 helicase. In Chapter 4, I demonstrated, for the first time, that G4 DNA structures can undergo phase separation. By utilizing immunofluorescence microscopy and ChIP-seq analysis, I observed that phase separation modulates the stabilities of G4 structures in vitro and in cells. This discovery provides new insights into factors that modulate the formation and stabilities of G4 structures in cells. In Chapter 5, I identified novel small GTPases as biomarkers of radioresistance in breast cancer cells through a multiple-reaction monitoring-based targeted proteomics analysis. The study revealed ARFRP1 as a novel radioresistance biomarker in breast cancer cells, where its downregulation promotes radioresistance. In conclusion, this dissertation presents novel proteomic approaches for the identification of G4-binding proteins, understanding G4 stability modulation, and the discovery of potential biomarkers in breast cancer cells. These findings enhance our understanding of G4 biology and offer new avenues for therapeutic interventions and biomarker-driven cancer research.

Post-translational modifications (PTMs) constitute an evolutionally conserved and ubiquitous mechanism for regulating protein structure and function. The first part of this dissertation focuses on the discovery of an epitranscriptomic mechanism of regulation N-terminal methyltransferase 1 (NTMT1) expression, and the identification of novel substrates of α-N-methylation and histidine methylation. The goal of the second half of this dissertation is to discover nucleotide-binding proteins (i.e. small GTPases and kinases) as drivers and suppressors for cancer metastasis and acquired radioresistance. In Chapter Two, we discovered that NTMT1 expression is regulated by reader/writer/eraser proteins of N6-methyladenosine (m6A). We further identified Mortality Factor 4 Like 1 (MRG15) as a novel α-N-methylated protein. We subsequently demonstrated that NTMT1 is responsible for α-N-methylation of MRG15, and this methylation is also regulated through an m6A-based epitranscriptomic mechanism. In Chapter Three, we interrogated a series of publicly available mass spectrometry datasets with MaxQuant to identify novel protein substrates for α-N-methylation. Our results uncovered 219 instances of N-terminal methylation in 196 proteins. We then identified, through affinity purification and mass spectrometry analysis, that Vesicle-associated membrane protein 4 (VAMP4) is α-N-methylated. We subsequently confirmed that NTMT1 is the primary enzyme responsible for the N-terminal methylation of VAMP4. In Chapter Four, we analyzed publicly available mass spectrometry datasets to identify novel protein substrates for histidine methylation. Using this method, we uncovered 33 instances of histidine methylation among 26 proteins. We subsequently used affinity purification and mass spectrometry analysis to confirm the histidine methylation of Pre-mRNA-splicing factor RBM22 (RBM22). In Chapter Five, we employed stable isotopic labeling by amino acids in cell culture (SILAC) and parallel-reaction monitoring (PRM) to monitor the differential expression of kinases in MCF-7 and MDA-MB-231 breast cancer cells and their corresponding radioresistant C6 and C5 clones. Using this method, we were able to identify and quantify the relative expression levels of 300 and 281 kinases in C5/MDA-MB-231 and C6/MCF-7 pairs of breast cancer cells, respectively. We further identified transcription initiation factor TFIID subunit 9 (TAF9) as a driver of radioresistance in breast cancer cell lines. In Chapter Six, we employed a proteomic method, based on multiple-reaction monitoring (MRM), for quantitative profiling of GTP-binding proteins using isotope-coded GTP probes. After probe labeling, tryptic digestion, and affinity enrichment of labeled peptides, the expression level differences of GTP-binding proteins in two matched pairs of primary/metastatic melanoma cell lines (WM-115/WM-266-4 and IGR-39/IGR-37) were investigated with LC-MRM analysis. Among the most upregulated proteins, adenylate kinase 4 (AK4) was identified as a potential driver of melanoma metastasis. We further demonstrated that AK4 expression is necessary for melanoma cell invasion and migration.

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.

Endogenous and environmental agents can cause DNA damage in cells. A number of alkylating agents introduce modifications into DNA bases that can be cytotoxic and mutagenic to the cell. N2-alkyl-2’-deoxyguanosine (dG) lesions are a primary type of DNA damage products resulting from alkylation that have been identified in the genome. While most of the DNA damage can be removed through different types of DNA repair pathways, any unrepaired DNA lesions may obstruct replication and transcription processes and further trigger genome instability. However, there is limited understanding regarding the impact of N2-alkyl-dG lesions in cellular processes and the functions of translesion synthesis (TLS) polymerases in modulating genome instability.In Chapter 2, we synthesized ODNs harboring N2-alkyldG lesions and incorporated them into plasmid vectors. By performing in vivo replication assay in wild-type and TLS polymerase-deficient E. coli cells, we found that N2-Et-dG is slightly less blocking to DNA replication than the two isomeric N2-nBu-dG lesions, which display very similar replication bypass efficiencies. Additionally, Pol II and, to a lesser degree, Pol IV and Pol V are required for the efficient bypass of the N2-alkyl-dG adducts, and none of these lesions were mutagenic. In Chapter 3, we used two independent approaches (proximity labeling and affinity pull-down) followed by LC-MS/MS analysis to profile Pol κ-interacting proteins at the proteome-wide level. Our result revealed that Pol κ interacts with DDX23, and Pol κ ChIP-Seq analyses indicated that Pol κ is enriched at R-loop structure loci in chromatin. In addition, Pol κ recruits DDX23 to R-loop sites in chromatin and promotes unwinding of R-loop structure. We also observed an augmented accumulation of R-loops in Pol κ- and DDX23-deficient cells. Together, we discovered an interaction between Pol κ and DDX23, and revealed that the interaction facilitates resolution of R-loops in cells. In Chapter 4, we demonstrated N2-alkyl-dG adducts induce R-loop structure accumulation on human chromatin by conducting immunofluorescence microscopy and sp-KAS-seq analysis. R-ChIP-qPCR assay further illustrates R-loop structures formed on lesion insertion sites in lesion-bearing episomal plasmids and cellular transcription assay results showed the lesion-induced R-loops strongly impede transcription. We also observed the accumulation of double-strand breaks (DSBs) after N2-alkyl-dG incorporation in DDX23-deficient cells. Together, we conclude that N2-alkyl-dG adducts trigger R-loop accumulation and further induce genome instability in human chromatin. Together, the research described in this dissertation reveals the adverse biological consequences of N2-alkyl-dG lesions and provides a much better understanding of the functions of TLS polymerases in modulating replicative bypass of N2-alkyl-dG lesions and maintaining genome stability.