Book Summary:
Mass spectrometry has become the analytical technique of choice for proteomics, due to its sensitivity and high-throughput nature. In this dissertation, I report on the development of several isotope-assisted proteomic techniques and their application to two multicellular eukaryotes, an animal, Electrophorus electricus and a plant, Arabidopsis thaliana. Electrophorus electricus, the electric eel, is the most strongly electrogenic organism known. As a result of its highly specialized lifestyle, Electrophorus has evolved three electric organs all of myogenic origin. The main organ is responsible for the strong discharge that has earned the eel its name while Sach's and Hunter's organs are involved in communication and environmental sensing. Few studies have addressed differences between these organs at the protein level. In order to expand our understanding of the relative differences, I compared microsomal fractions from the main and Sach's electric organs using 18O-isotopical labeling. Multiple channels, pumps, and GTP-binding proteins showed relative differences between these two organs in excess of two-fold. The plasma membrane serves as the boundary between the cell interior and exterior and is the location of extensive signal and energy transduction. Because of its integral role in cell biology, several proteomic investigations have catalogued proteins localized to the plasma membrane. Prior plasma membrane studies in the model higher plant, Arabidopsis thaliana, have either lacked quantitation to address contamination issues or alternatively, performed poorly with integral proteins. Using 18O-isotopic labeling and mass spectrometry, I assessed the issue of contamination in plasma membrane preparations. Metabolic labeling with 15N and concomitant proteomic analysis has several advantages over in vitro isotopic labeling techniques, and could become a powerful analytical tool in plant biology. Although 15N-metabolic labeling has been applied to prokaryotes, yeast and metazoans, there are no published reports of metabolic labeling of plants in a proteomic context. In this thesis, 15N-nitrate was provided in growth medium for Arabidopsis to generate a 15N labeled proteome. New informatic tools were applied to reduce false positives and false negative identifications. Quantitative tools were also developed and used to measure relative changes between natural abundance and 15N-metabolically labeled samples. |