Methods for Identification and Quantitation of Small Molecules and Peptides in Bioenergy, Metabolism, and Proteome Research.

摘要

This dissertation considers the development of methods and hardware for identification and quantitation of small molecule and peptide analytes. Specifically, (1) LC-MS, GC-MS, and LC-UV techniques for the identification and quantitation of lignin-derived aromatics and the application of those techniques, (2) LC-MS analyses of low-abundance lipids with special emphasis on Coenzyme Q metabolites, (3) the development of two NeuCode quantitation methods, (4) methods for the improvement of precursor ion selection in proteomic experiments, and (5) the improvement of proteome digest separations by custom-designed and manufactured column thermostats are explained in detail in the following chapters. Chapter 1 provides a brief overview and some background on the measurement of molecules by chromatography coupled to either a mass spectrometer or a spectrophotometer. The factors that dictate the choice of technique are discussed, as are determinants of measurement precision and accuracy. Special attention is given to factors relevant to the research projects discussed in this dissertation. Chapters 2 and 3 discuss the development and assessment of three novel NeuCode quantitation methods for organic acids and peptide digests by LC-MS and GC-MS. Chapter 4 describes segmenting the mass spectrometer scan range for optimization of precursor selection in proteomic studies. Chapter 5 reports on the design, manufacture, and characterization of custom column ovens for improved chromatographic protein digest separations. Chapter 6 describes the applications of LC-MS, LC-UV, and GC-MS methods I developed to the depolymerization of lignin to value-added aromatics, the quantification of fermentation inhibitors during bioethanol production, and the characterization of proteins involved in Coenzyme Q biosynthesis.