Date of Award
Doctor of Philosophy (PhD)
Biochemistry & Molecular Biophysics
Benjamin A. Garcia
Epigenetic and epitranscriptomic mechanisms play critical roles in regulating transcription and translation, among other cellular functions. One of the most important ways this is performed is through chemical modification of proteins, such as histones, and nucleic acids, such as RNA. These modifications dramatically increase the diversity of unique molecules, resulting in well over 1,000 histone proteoforms and over 150 unique modified RNA nucleotides. The complexity of these systems makes them challenging to interrogate and obtaining all meaningful information in a single experiment remains impossible. In this dissertation, I introduce novel mass spectrometry approaches to analyzing modified histones and modified RNA nucleosides and apply them to biological systems. Specifically, I address middle-down proteomics of histone tails, which is sparsely performed due to technical challenges. I introduce a simplified method that is robust and reliable and can quantitate both middle-down and bottom-up histone peptides simultaneously. Histone proteomics was applied to mitochondrial mutations, which enabled the identification of mitochondrially regulated nuclear gene expression. RNA modifications and their functions also remain enigmatic due to the availability and power of tools used for studies. Although the most common mass spectrometry methods for RNA modification analysis lose sequence information due to digestion, they outperform sequencing in every other metric. For this reason, MS analysis of RNA nucleosides can be of very high value. Methods were designed to address potential pitfalls in sample preparation and reduce biases in LC-MS/MS based detections. The methods were applied to different classes of RNA and provided insight into translation mechanisms and tRNA processing regulation. Together, these studies demonstrate advances in techniques used to investigate fine regulation of transcription and translation, and their applications yielded both novel regulatory relationships and insights into yet unstudied molecular functions.
Janssen, Kevin, "Mass Spectrometry Methods For Enhanced Analysis Of Epigenetic And Epitranscriptomic Modifications" (2020). Publicly Accessible Penn Dissertations. 4012.