UC Riverside

Recent advances in mass spectrometry instrumentation and sample preparation methods have enabled robust identification and quantification of proteins at the entire proteome level. The focus of this dissertation is placed on two groups of proteins, the TBC domain-containing proteins as well as epitranscriptomic reader, writer and eraser (RWE) proteins. In Chapter 2, I utilized a shotgun quantitative proteomic method to assess, at the global proteome scale, differential protein expression in a matched pair of primary/metastatic melanoma cell lines (i.e. WM-115/WM-266-4). I found TBC1D7 may play a role in melanoma cell invasion. In Chapter 3, I established a liquid chromatography–parallel-reaction monitoring (LC-PRM) method for high-throughput profiling of approximately 150 epitranscriptomic RWE proteins. I employed this LC-PRM method coupled with stable isotope labeling by amino acids in cell culture (SILAC) to examine the differences in expression levels of the proteins in two matched pairs of radioresistant/wild type (MDA-MB-231/C5 and MCF-7/C6) breast cancer cells. This method allows for the quantifications of 70% and 65% of the epitranscriptomic RWE proteome. Among them, TRMT1 (an m2,2G writer) may assume a crucial role in enhancing breast cancer radioresistance. In Chapter 4, I further applied this LC-PRM method to assess the expression of epitranscriptomic RWE proteins in modulating colorectal cancer (CRC) metastasis. I was able to quantify 74% of the epitranscriptomic RWE proteome; among them, 48 and 5 were up- and down-regulated by over 1.5-fold in metastatic SW620 relative to primary SW480 CRC cells, respectively. In Chapter 5, I modified the LC-PRM method by employing a mixture of 48 stable isotope-labeled (SIL) peptides representing RWE proteins as internal or surrogate standards. I utilized this method to explore potential crosstalk between N6-methyladenosine (m6A) and other modified ribonucleosides by assessing the epitranscriptomic RWE proteome in ALKBH5-/-, FTO-/-, METTL3-/- cells, and their isogenic parental HEK293T. NOP2, PUS3, TGS1 and RBMX were altered by more than 1.5-fold in the opposite directions in ALKBH5-/- and METTL3-/- cells relative to isogenic HEK293T cells. Together, the research described in this dissertation documented the power of quantitative proteomics in revealing new functions of cellular proteins in modulating cancer metastasis, radioresistance, and the epitranscriptome.