Date of Graduation
Eberly College of Arts and Sciences
Fred L. King.
In this document methods for the analysis of proteins and small molecules by Electrospray Ionization Mass Spectrometry (ESI-MS) are discussed. Section I contains an introduction to ESI-MS, including a brief history of mass spectrometry, a discussion of the current theories developed to explain the mechanisms at work during the electrospray process, and an explanation of Ion Trap Mass Spectrometry (IT-MS), the type of mass analyzer used for the studies outlined in subsequent chapters.;Section II is devoted to the analysis of proteins by ESI-MS. Chapter 2 gives an overview of protein structure and reviews previous work in the area of protein structural analysis by ESI-MS. Chapter 3 summarizes experimental work done in our laboratory with regard to the influence of instrumental parameters on the Charge State Distribution (CSD) of the heme proteins cytochrome c and myoglobin, and discusses a method for monitoring conformational changes of proteins by calculating an Average Observed Charge (AOC) for a given spectrum.;Chapter 4 is also devoted to protein conformational analysis by ESI-MS. This work outlines the changes to the CSD and AOC when an organic sheath liquid is infused to the ion source and co-electrosprayed with the aqueous solution containing the proteins. In Chapter 5 Collision Induced Dissociation (CID) is utilized to determine the energy required to liberate the prosthetic heme group from the various charge states of myoglobin, in which the heme is not covalently associated with the polypeptide portion of the molecule, and cytochrome c in which the heme group is tethered to the polypeptide by two covalent linkages. Heme ion intensity is monitored as a function of applied collision energy, and the results are displayed graphically in dissociation plots. From the plots it can be seen that less energy is required to liberate the heme group from the higher charge states. Additionally, dissociation plots for like charge states of the two proteins are directly compared. From this comparison it is observed that significantly more energy is required to dissociate the heme group form cytochrome c than from myoglobin, consistent with the stronger heme:protein association due to the presence of the covalent bonds in cytochrome c that are absent in myoglobin.;Chapter 6 details multiple-stage mass analysis (MSn) to determine the fragmentation pathways followed by the heme groups liberated by CID from myoglobin and cytochrome c. A mass difference of 1 AMU is observed for the heme groups from the two proteins, though the heme group is of the same type. The mass difference appears to create a bias toward one of two competing fragmentation pathways that are observed to some degree in the heme groups of both proteins.;Section III outlines work in the analysis of small molecules by ESI-MS. Chapter 7 describes the identification of a urinary metabolite of nicotinylalanine by LC/ESI-MS. The identification is made by comparison of data obtained from LC retention time analysis, and single-stage and multiple-stage mass analysis of the urinary metabolite and the synthetic material. A curriculum vita (CV) for the author follows Section III, and References conclude this document.
Pratt, Gregg A., "Electrospray ionization mass spectrometry for the analysis of proteins and small molecules" (2000). Graduate Theses, Dissertations, and Problem Reports. 1238.