| Mass spectrometry has emerged as a precise and sensitive method for analysis of biological molecules, especially proteins. Top-down protein mass spectrometry uses intact and fragment masses of proteins to identify gene products and their respective post-translational modifications. This thesis reports improved fragmentation, gas-phase isolation, and data processing methods for top-down mass spectrometry using Fourier transform mass spectrometry.;The general aim of the method developments are (1) to improve the ability to identify and pinpoint post-translational modifications, and (2) to enable high resolution intact protein 1H/2H exchange experiment. These goals stem from the specific aims of (1) characterization of SOD1 post-translational modification occurring in vivo in amyotrophic lateral sclerosis patients and controls, and (2) the effects of these modifications upon SOD1 quarternary structure and dynamics.This thesis reports the development of (1) a novel and efficient gas-phase molecular fragmentation technique termed funnel-skimmer dissociation (Chapter 1), (2) an extended protein database and novel search engine capable of processing top-down MS2 data, Mascot TD (Chapter 1), (3) the first time gas-phase separation of multiple charge states of a modified protein isoform was used together with electron capture dissociation (Chapter 2), (4) A novel algorithm to generate theoretical spectra developed in our lab was tested for intact proteins and ultra-high resolution peptide spectra (Chapter 3).;The fragmentation and data processing improvements presented here extended the upper mass limit of top-down protein identification from 200 to 660 kDa and enabled the identification of post-translational modification of human Cu/Zn superoxide dismutase caused by its enzymatic reaction product, hydrogen peroxide (Chapter 4).;This thesis also experimentally tested, for the first time, the relationship of magnetic field strength and Fourier transform ion cyclotron resonance mass spectrometry performance using three instruments with the same designs but different magnetic fields of 4.7, 7 and 9.4 tesla (Chapter 5). The predicted "transformative" effects of magnetic field were indeed observed experimentally.;In summary, this thesis demonstrates that combination of Fourier transform ion cyclotron resonance mass spectrometry and related data analysis methods are precise, sensitive and specific tools that can be applied for reliable characterization of post-translational modifications and for identification of any size of intact proteins. |
