| Since the completion of the Human Genome Project and subsequent development of genomic and proteomic databases, proteomics research has experienced explosive growth. Proteomics is the venture to identify and characterize proteins encoded by genes, including post-translational modifications and alternatively-spliced forms. Electrospray ionization-mass spectrometry has become a key technology for proteomics measurements owing to the afforded sensitivity and molecular specificity of the technique. Moreover, in this dissertation it is shown that the ability of Fourier transform-ion cyclotron resonance mass spectrometry to analyze large numbers of analytes (known as the peak capacity) enables complex mixture analysis. Mass spectrometry-based proteomics measurements to date in large part have focused on the identification of proteins, including post-translational modifications. Increasingly, mass spectrometry based proteomics methods for absolute and relative quantification are emerging. Herein, normalization strategies utilizing synthetic exogenous peptides with masses that do not overlap with endogenous peptides, allowing for the assessment of mass measurement accuracy and retention time reproducibility, are presented. In combination, the internal standards and normalization strategies afford accurate and reproducible protein quantification. Little research has been published to date that explores the fundamental aspects of quantitative proteomics. The work presented in this dissertation examines fundamental aspects, specifically, the role physical (post-excite radius, axial confinement fields, signal decay rate, temperature) and chemical (hydrophobicity) characteristics have on accurate protein quantification by electrospray ionization-Fourier transform-ion cyclotron resonance mass spectrometry. Examination of the instrumental parameters (post-excite radius, excitation voltages, trapping voltages, excitation waveforms) that lend to confident identification and accurate quantification of proteins in complex mixtures enables future quantitative proteomics measurements. Moreover, the understanding and development of strategies that increase ion abundance (hydrophobicity), and subsequently lower the limit of detection, will facilitate future studies of low abundance proteins. |
