Development of Proteomic Strategies to Elucidate Disease Processes in Cancer and Neurological Disorders

Advances in instrumentation and data analysis software have allowed mass spectrometry to become perhaps the most important tool in large scale proteomic studies. Using optimized workflows, it is possible to identify thousands of proteins and protein post-translational modifications in a single mass spectrometry (MS) run from a variety of sample sources, including immortalized cell lines, tissues, and biofluids. However, protein phosphorylation still presents a challenge for analysis because its low abundance and dynamic nature necessitates additional sample preparation steps and special considerations that must be taken into account when quantifying phosphorylation events. Even more difficult is dissection of intricate phosphorylation signaling pathways, which are involved in healthy cellular processes as well as disease pathogenesis. Protein post-translational modifications aside, identification of low abundance proteins from certain sample types can also be problematic. A good example of this is the so-called secretome, or the proteins that are secreted by cells in response to various stimuli. Secreted proteins are released in small amounts into large volumes of complex biofluids or cell culture media, making it difficult to recover these proteins for MS analysis This dissertation discusses strategies for identifying segments of both the phosphoproteome and the secretome. Chapter one is an overview of current phosphoproteomic techniques while chapters two and three highlight the work that has been done to identify the direct substrates of the ABL and CDKL5 kinases, whose dysregulation leads to aberrant phosphorylation signaling and the development of cancer or severe infantile epilepsy, respectively. Chapter four details the development of a novel nanopolymer-based reagent for capture of the secretome from Helicobacter pylori, a pathogen that has been linked to peptic ulcer formation and several types of cancer

Viral proteogenomic and expression profiling during productive replication of a skin-tropic herpesvirus in the natural host.

Efficient transmission of herpesviruses is essential for dissemination in host populations; however, little is known about the viral genes that mediate transmission, mostly due to a lack of natural virus-host model systems. Marek's disease is a devastating herpesviral disease of chickens caused by Marek's disease virus (MDV) and an excellent natural model to study skin-tropic herpesviruses and transmission. Like varicella zoster virus that causes chicken pox in humans, the only site where infectious cell-free MD virions are efficiently produced is in epithelial skin cells, a requirement for host-to-host transmission. Here, we enriched for heavily infected feather follicle epithelial skin cells of live chickens to measure both viral transcription and protein expression using combined short- and long-read RNA sequencing and LC/MS-MS bottom-up proteomics. Enrichment produced a previously unseen breadth and depth of viral peptide sequencing. We confirmed protein translation for 84 viral genes at high confidence (1% FDR) and correlated relative protein abundance with RNA expression levels. Using a proteogenomic approach, we confirmed translation of most well-characterized spliced viral transcripts and identified a novel, abundant isoform of the 14 kDa transcript family via IsoSeq transcripts, short-read intron-spanning sequencing reads, and a high-quality junction-spanning peptide identification. We identified peptides representing alternative start codon usage in several genes and putative novel microORFs at the 5' ends of two core herpesviral genes, pUL47 and ICP4, along with strong evidence of independent transcription and translation of the capsid scaffold protein pUL26.5. Using a natural animal host model system to examine viral gene expression provides a robust, efficient, and meaningful way of validating results gathered from cell culture systems