Global and targeted proteomics in Arabidopsis thaliana: A study of secondary metabolism and phytohormone signaling
Slade Jr, William O.
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Proteomics is defined as a tool to explore how proteins control and regulate important molecular and physiological processes. Further, peptide-centric approaches, or bottom-up methods, provide more comprehensive coverage of a proteome compared to whole-protein approaches. This body of work assesses the technical feasibility of several bottom-up proteomics technologies applied to Arabidopsis thaliana, including gel-based methods, those that require peptide derivitization, and those that do not. Selected-reaction monitoring (SRM) for targeted proteomics, and data-independent acquisition (MSE) was also evaluated. In addition to assessing the capabilities of these technologies, we then applied them to the context of uncovering new insights into the flavonoid biosynthetic pathway and the auxin and ethylene signaling pathways. Chapter one provides background information related to secondary metabolism, phytohormone signaling, and the status of proteomics in plants. In Chapter 2 and Appendix A, we establish the methodology to apply traditional and DiGE-based 2D-GE strategies to global proteomics in Arabidopsis. Our results suggest that while 2D-GE is applicable to Arabidopsis, there are practical and conceptual limitations that must be understood. Further, our results suggest that pertubations in the flavonoid pathway do not affect the abundance of proteins in Arabidopsis seedlings, roots, or flowers that can be studied using 2D-GE and DiGE. Additionally, we demonstrated the first parallel comparison of the effects of auxin and ethylene on the Arabidopsis root proteome and observed no overlap among the proteins regulated by the two phytohormones, at least for the most abundant proteins observed by 2D-GE. Chapter 3 explores the efficacy of selected reaction monitoring for relative peptide quantification in Arabidopsis roots. Our results suggest that while the technology parallels application in yeast and humans, there are substantial analytical challenges that much be addressed. In Chapter 4 we explore the MSE data acquisition scheme for global proteomics in Arabidopsis. We observe that treatment with exogenous auxin affects the abundance of many proteins representing diverse biological processes. Interestingly, we observe minimal overlap among genes and proteins regulated by exogenous auxin. Appendix B explores the efficacy of iTRAQ labeling for relative peptide quantification in Arabidopsis roots.
- Doctoral Dissertations