Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references. Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (p. 154-168).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 143-156).

Field asymmetric waveform ion mobility spectrometry (FAIMS) is a gas phase atmospheric pressure separation technique that exploits the difference in the mobility of ions in alternating low and high electric fields as they are carried between two electrodes. In this thesis, a miniaturised FAIMS separation step has been applied to increase selectivity, enhance sensitivity and improve the quality of mass spectral data for rapid, high-throughput protein and peptide analysis. In Chapter 2, charge state separations were used to generate pseudo-peptide mass fingerprint data by FAIMS-MS, permitting confident protein identification using ESI sample introduction as an alternative to MALDI-TOF-MS methods. In addition, pre-cursor ions were targeted prior to MS/MS analysis. Chapter 3 describes the analysis of intact proteins by miniaturised FAIMS-MS. Multiple charge states of intact proteins were separated on the basis of differences in differential mobility. Higher charge states were found to be transmitted at similar CVs suggesting that the miniaturised FAIMS device was separating ions on the basis of 3D structure. In addition, multiple species could be observed at the same m/z suggesting the presence of different protein conformers. In Chapter 4, miniaturised FAIMS was used to select ions on the basis of differential mobility prior to in-source collision-induced dissociation CID, LC and ToF-MS analysis for qualitative and quantitative analysis of peptides mixtures. This was applied to the analysis of co-eluting model peptides and tryptic peptides derived from human plasma proteins, allowing precursor ion selection and CID to yield product ion data suitable for peptide identification via database searching.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.

Dans le but de déterminer l'influence du groupement trifluorométhyle sur les propriétés physico-chimiques et biologiques de peptides fluorés, nous avons désiré synthétiser des peptides incorporant un aminoacide α-trifluorométhylé.Chaque énantiomère de l'α-Tfm-Alanine a été synthétisé de manière énantiomériquement pure et à grande échelle selon une voie synthétique efficace. Le groupement trifluorométhyle placé en position α désactivant fortement sa fonction amine, son couplage a nécessité des conditions d'activation puissantes à l'aide des anhydrides mixtes.La variation de l'hydrophobie de peptides fluorés par une méthode analytique basée sur la mesure d'indices dérivés des temps de rétention par RP-HPLC a permis de mettre en évidence l'influence considérable du groupement trifluorométhyle sur l'augmentation de l'hydrophobie.Les interactions peptides fluorés/peptides ont été étudiées dans le cadre de l'inhibition de l'agrégation du peptide Aβ42 responsable du dépôt de plaques amyloïdes chez les patients atteints de la maladie d'Alzheimer. Les premiers résultats montrent un ralentissement de l'agrégation de Aβ42 par un peptide fluoré.La digestion enzymatique par la pepsine d'un tétrapeptide fluoré indique un ralentissement considérable de sa vitesse d'hydrolyse par rapport au peptide incorporant un résidu Alanine. L'utilisation de la méthode de détection sensible par RMN 19F appelée 3-FABS a permis de mettre en évidence la reconnaissance et le clivage d'un peptide fluoré par la trypsine, caractéristiques d'un bon substrat. / In order to determine the impact of the trifluoromethyl group on the physico-chemical and biological properties of fluorinated peptides, we have decided to synthesize peptides incorporating α-trifluoromethyl amino-acids.Each enantiomer of α-Tfm-Alanine was prepared in an enantiopure form and in a large scale. The trifluoromethyle group placed in the α position deactivates its amine function so that its coupling needs harsh activation conditions to be achieved with mixed anhydride.The determination of the hydrophobicity of fluorinated peptides with an analytical method based on the measurement of indexes derived from retention times by RP-HPLC showed the dramatic influence of the trifluorométhyl group on the increase of the hydrophobicity.Fluorinated peptides/peptides interactions were studied for the inhibition of the aggregation of Aβ42 in patients suffering from Alzheimer's disease. The first results indicate a reduction of Aβ42 aggregation by a fluorinated peptide.Pepsine digestion of a fluorinated tetrapeptide showed a dramatic reduction of the rate of its cleavage in comparision with the peptide incorporating an alanine residu. The use of the sensitive 3-FABS 19F NMR detection method showed the recognition and the cleavage of a fluorinated peptide by trypsin which definites it as a substrate for trypsin.

In this thesis, improved techniques for biomolecule analysis using capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and hyphenations between those have been presented.A pre-concentration method which is possible to apply in both techniques, has also been investigated. In this work the off-line MS mode has been used either in the form of fractionation (Paper I) or by incorporating the MALDI target in the CE separation system (Paper II).In Paper I, a protocol for CE-MALDI analysis of cyanogen bromide digested bacteriorhodopsin (BR) peptides as model integral membrane protein peptides were established. Also, an improved protocol for partially automated manufacturing of a concentration MALDI-target plate is presented. The design of the targets was suitable for the fractions from the CE. A novel technique for the integration of CE to MALDI-MS using a closed-open-closed system is presented in Paper II, where the open part is a micro canal functioning as a MALDI target window. A protein separation was obtained and detected with MALDI-MS analysis in the micro canal. A method has been developed for detection of monosaccharides originating from hydrolysis of a single wood fiber performed in a micro channel, with an incorporated electromigration pre-concentration step preceding CE analysis in Paper III. The pre-concentration showed to be highly complex due to the fact that several parameters are included that affecting each other. In Paper IV a protocol using enzymatic digestion, MALDI-TOF-MS and CE with laser induced fluorescence (LIF) detection for the investigation of the degree of substitution of fluorescein isothiocyanate (FITC) to bovine serum albumin (BSA), as a contact allergen model system for protein-hapten binding in the skin, is presented. The intention of a further CE-MALDI hyphenation has been considered during the work. In Paper V 2,6-dihydroxyacetophenone (DHAP) was investigated, showing promising MALDI-MS matrix properties for hydrophobic proteins and peptides. 2,5-dihydroxybenzoic acid (DHB) was undoubtedly the better matrix for the hydrophilic proteins, but its performance for the larger and hydrophobic peptides was not optimal. Consequently, DHAP can be used as a compliment matrix for improved analysis of hydrophobic analytes. / QC 20101214