Alzheimer’s is a widespread neurodegenerative disease, growing larger and larger in the world. Once developed, the disease has no cure. To this day, there is only mitigating drugs. To be able to start this treatment rapidly, a method to distinguish healthy individuals from prospective Alzheimer’s diseased needs to be developed. Cerebrospinal fluid is thought to contribute to the development of such method, through its close substitution of fluids, molecules and proteins from the brain. It may provide a progressive marker of the disease, a substance differently expressed in the healthy and diseased; a biomarker. The aim of the present study was to develop and evaluate a stable method for degradation and analysis of peptides and proteins in the cerebrospinal fluid using mass spectrometry techniques, such as selective reaction monitoring. Mass spectrometry is often used after a first dimension separation with liquid chromatography. Three degradation methods were evaluated, resulting in a protocol with the detergent DOC being the most beneficial. Tryptic peptides occurred in a concentration of 10 % w/v due to the SDS-PAGE gels and database searches concomitantly. The elution pattern from the liquid chromatography enables a narrow selection in the sensitivity for each peptide. Chromatographic preferences such as column, hydrophobicity and time span was determined, and unwanted peptides filtered away. A specific protein, the amyloid precursor protein APP, is thought to play a significant part in the development of Alzheimer’s disease. The protein is located in the neurons, cleaved and processed to produce the neurodegenerating plaques found in the brain at the diseased. Three isoforms are found in the neurons, APP695, APP751 and APP770. When cleaved, a shorter soluble tryptic peptide is generated from all APP isoforms. This was the target for the current study, as a potential progressive biomarker. The method developed was able to separate and distinguish the soluble APP751 isoform, but not the APP695 or APP 770 isoforms, most probably due to glycosylations of the two resembling isoforms.

A new laser ablation mechanism of liquid water based on recent insights into its hydrogen bond dynamics has been studied and several applications of the ablation demonstrated. The mechanism, termed as Impulsive Heat Deposition through Vibrational Excitations (IHDVE), is based on the ability of the hydrogen bond network of water to rapidly thermalize vibrational O-H stretch excitations on a time scale of several picoseconds even for excitation intensities that are large enough to bring excited volumes far into the supercritical region. In this way, by using vibrationally resonant picosecond infrared laser pulses with sufficient energy, it is possible to drive ultrafast phase transitions in the excited water volume leading to a rapid and efficient ablation process of water and water rich targets with minimum perturbation of solute molecules of interest. The physics behind the IHDVE ablation process is outlined and the benefits of the IHDVE ablation are demonstrated for two important applications of tissue cutting and mass spectrometry of biomolecules. Finally, the development of two high power infrared laser systems suitable for the practical implementation of IHDVE is presented.

High-pressure mass spectrometry has been employed to investigate hydrogen and sodium bound ion-molecule complexes in the gas phase. Insight into the structure and reactivity of ion-molecule complexes has been gained by examining simple mono-ketones of butanone and 2-pentanone and more complex β-diketones of 2,4-pentanedione, 1,1,1-trifluoro-2,4-pentanedione and 1,1,1,5,5,5-hexafluoro-2,4-pentanedione. The effect of fluorinating 2,4-pentanedione exhibits experimental trends that are not representative of current electronic structure calculations . A novel method of sodium ion production has been developed using sodium metal, allowing for equilibrium measurements below 150 °C. Sodium containing ion-molecule complexes have been investigated using the new method of sodium ion production.

High-pressure mass spectrometry has been employed to investigate hydrogen and sodium bound ion-molecule complexes in the gas phase. Insight into the structure and reactivity of ion-molecule complexes has been gained by examining simple mono-ketones of butanone and 2-pentanone and more complex β-diketones of 2,4-pentanedione, 1,1,1-trifluoro-2,4-pentanedione and 1,1,1,5,5,5-hexafluoro-2,4-pentanedione. The effect of fluorinating 2,4-pentanedione exhibits experimental trends that are not representative of current electronic structure calculations . A novel method of sodium ion production has been developed using sodium metal, allowing for equilibrium measurements below 150 °C. Sodium containing ion-molecule complexes have been investigated using the new method of sodium ion production.

Determination of lead and iodine species by capillary electrophoresis inductively coupled plasma mass spectrometry.

µL

none

none

Separations coupled to mass spectrometry (MS) are widely used for large-scale protein identification in order to reduce the adverse effects of analyte ion suppression, increase the dynamic range, and as a deconvolution technique for complex datasets typical of cellular protein complements. In this work, matrix assisted laser desorption-ionization is coupled with ion mobility (IM) separation for the analysis of biological molecules. The utility of liquid-phase separations coupled to MS lies in the orthogonality of the two separation dimensions for all analytes. The data presented in this work illustrates that IM-MS relies on the correlation between separation dimensions for different classes (either structural or chemical) of analyte ions to obtain a useful separation. For example, for a series of peptide ions of increasing mass-to-charge (m/z) a plot drift time in the IM drift cell vs. m/z increases in a near-linear fashion, but DNA or lipids having similar m/z values will have very different IM drift time-m/z relationships, thus drift time vs. m/z can be used as a qualitative tool for compound class identification. In addition, IM-MS is applied to the analysis of large peptide datasets in order to determine the peak capacity of the method for bottom-up experiments in proteomics, and it is found that IM separation increases the peak capacity of an MS-only experiment by a factor of 5-10. The population density of the appearance area for peptides is further characterized in terms of the gas-phase structural propensities for tryptic peptide ions. It is found that a small percentage (~3%) of peptide sequences form extended (i.e., helical or β-sheet type) structures in the gas-phase, thus influencing the overall appearance area for peptide ions. Furthermore, the ability of IM-MS to screen for the presence of phosphopeptides is characterized, and it is found that post translationally modified peptides populate the bottom one-half to one-third of the total appearance area for peptide ions. In general, the data presented in this work indicates that IM-MS offers dynamic range and deconvolution capabilities comparable to liquid-phase separation techniques coupled to MS on a time scale (ms) that is fully compatible to current MS, including TOF-MS, technology.

Thesis (Ph. D.)--Open University. BLDSC no. DX225509.