Electrospray Ionization (ESI) is a technique that permits the soft ionization of large proteins and biomolecules without fragmenting them, which allows them to be characterized via Mass Spectrometry (MS). It has the potential of permitting the identification of transient intermediate products in biological processes in situ, which would provide great insight to researchers in the growing fields of proteomics and metabolomics. However, this application presents a technical challenge in that most relevant biochemistry occurs in aqueous solutions with high salt content, which makes successful identification of analytes by ESI-MS difficult. This thesis presents the design, fabrication, and characterization of a microfabricated dialysis module that could alleviate this issue by desalting samples inline between sampling and electrospraying interfaces. Its small volume (~10 nL) minimizes sample transit time and, thus, optimizes ESI-MS analysis temporal resolution. A preliminary analytical model of dialysis elucidates the key performance parameters and sets the guidelines for consideration in its design. The device is then microfabricated in a cleanroom environment using techniques that have been well established by the microelectronics industry such as E-beam evaporation and Reactive Ion Etching. The system efficiency is demonstrated experimentally by assessing its salt removal effectiveness as a function of sample residence time. Mass spectrometry analyses of proteins in solutions with high salt content further corroborate its performance.

Thesis (Ph.D.) -- McMaster University, 2001. / Includes bibliographical references. Also available via World Wide Web.

Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Jennifer S. Brodbelt. Vita. Includes bibliographical references. Also available from UMI.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 144-158).

Lignin, the second most abundant naturally occurring polymer found in plant cell wall has the potential of becoming an alternative source for the production of chemical synthons for the pharmaceuticals and other chemical industries. While much gain has been made towards the development of degradation methods to break down lignin, effective analytical methods are still required to rapidly and accurately identify the products of lignin breakdown experiments. The goal of this work was to develop mass spectrometric methods for the characterization of lignin oligomers based on the study of model lignin compounds. Unlike peptides and oligosaccharides, lignin model compounds that could serve as analytical standards for methods developments are not commercially available, and hence, the first project of this dissertation focused on the synthesis of lignin model compounds containing the β-O-4 bond. The priority was to synthesize compounds containing all important functionalities that reflect the structure of native lignin. By employing the known Aldol reaction, lignin oligomers containing the β-O-4 were synthesized. The synthesized β-O-4 lignin oligomers contained the characteristic functional groups of native β-O-4 lignin, that is, the phenolic functionality, the aryl glycerol β-O-4 aryl ether bond and the unsaturated side chain. The second project was aimed at developing alternative ionization methods for the characterization of lignin in the negative ion mode mass spectrometry. A chloride adduct ionization method was developed and used for characterizing and sequencing lignin oligomers. This method proved to be very useful in stabilizing the adduct ion in the full scan spectrum mode and also providing useful structural information upon tandem mass spectrometry. In the third project, a cationization technique was developed to unambiguously assign the sequence in which β-O-4 lignin oligomers are connected. A simple and easy to use sequencing chart was designed and could serve as a guide for predicting the sequence of larger lignin oligomers. This method offers an alternative approach for the characterization of lignin oligomers in the positive ion mode mass spectrometry. The fourth project focused on the ionization response of a new class of β-O-4 lignin compounds. β-O-4 compounds having the same skeletal backbone but different non-polar groups at the a-position were synthesized, and their ESI response studied. Results from this study show that a slight change in the structure of a β-O-4 lignin model compound can change the cationization response to several order of magnitude. Most importantly, this work for the first time has shown a direct correlation between lignin ionization response and lignin structure. The fifth project was aimed at studying the chromatographic behavior of the diastereomer pair in β-O-4 lignin model compounds. Using three commercially available HPLC columns, the chromatographic behavior and factors that affect the separation of the diastereomer pair of the β-O-4 lignin diastereomer on an HPLC column were studied. By performing tandem mass spectrometry on each of the diastereomers, a fragmentation mechanism was developed that could be used to unambiguous assign the configuration (erythro or threo) for the pair of diastereomer in a β-O-4 model. The results presented in this dissertation adds significant knowledge to the lignin mass spectrometry literature, and it offers new ionization techniques for the characterization of lignin oligomers, most importantly, an alternative approach for lignin analysis using adduct ionization mass spectrometry. The developed methods could easily be extended for the characterization of larger lignin oligomers.

No description available.

L'héparine (Hp) est un polysaccharide sulfaté appartenant à la famille des glycosaminoglycanes (GAGs), et est constitué d'unités de répétition disaccharidiques composées d’un acide hexauronique lié par une liaison α1→4 à un résidu hexosamine. La sulfatation de ce polysaccharide peut avoir lieu sur les positions 6-O ou N du glucosamine, mais également sur la position 2-O de l'acide hexauronique. En général, les GAGs sont O-liés aux chaînes latérales des protéoglycanes, et sont associés à un nombre important d'activités physiologiques, généralement reliées à leur interaction avec diverses protéines. Dans certains cas, cette interaction peut-être influencée par la liaison à ces complexes Hp/protéine d'ions métalliques naturels. Ceux-ci influencent l'affinité, la spécificité et la stabilité de ces complexes. En dépit de sa pertinence, le mécanisme par lequel un cation métallique module l'activité de l'héparine au sein des complexes Hp-protéine, reste largement méconnu.Un éventail de stratégies et d'outils ont été développés afin de faciliter la détermination des structures primaires des biomolécules par spectrométrie de masse en tandem (MS/MS). En effet, la caractérisation structurale de l'héparine sulfatée et de ses complexes métalliques a été soutenue par le développement de techniques de spectrométrie de masse. Dans certains cas, il a été observé que lors de l'activation par dissociation induite par collision (CID), certains de ces isomères d'héparine partageaient à peu près les mêmes schémas de fragmentation, compliquant de ce fait le processus d'identification de ces composés. Néanmoins, des études réalisées au LAMBE ont montré que la réactivité en phase gazeuse des ions métalliques pouvait aider à la différenciation d'isomères saccharidiques. Ces études peuvent être utiles non seulement du point de vue purement analytique, mais également parce que le comportement différent des isomères envers un métal donnée conduit à informations sur le processus d'interaction Hp/Métal mis en jeu. Cela peut s’avérer important pour interpréter les mécanismes biologiques mentionnées auparavant.Dugourd et al ont récemment rapporté les spectres optiques et les motifs de photodissociation de différents oligosaccharides sous irradiation UV. En terme de chemins de fragmentation, il a été observé que les spectres de photodissociation Ultraviolet (UVPD) apparaissaient être plus informatifs que la CID en raison de clivages à travers les cycles supplémentaires, qui fournissent une information sur la position du groupe sulfate. Le spectre optique des disaccharides sulfatés est caractérisé par une bande large et intense centrée vers 240 nm. Suite à ces résultats, nous nous sommes intéressés au couplage de la spectroscopie optique et des calculs théoriques pour les disaccharides d'héparine, et avons employé la spectroscopie UVPD afin d’obtenir des informations complémentaires sur les interactions de ces sucres avec les métaux.Comme décrit auparavant, les processus CID représentent un outil très précieux pour la caractérisation structurale des biomolécules. Motivés par des travaux antérieurs publiés par W. Hase et K. Song, nous avons collaboré avec les groupe de R. Spezia et T. Riera afin de parvenir à une meilleure compréhension du processus MS/MS des sucres et des peptides protonés. Les mécanismes CID de modèles simples, N-Formylalanylamide (HCO-Ala-NH2) et Galactose-6-Sulfate, ont été étudiés par des simulations de dynamique moléculaire QM+MM et des expériences MS/MS. Les objectifs de cette thèse étaient les suivants: i) explorer la possibilité d'utiliser le calcium métallique à des fins analytiques ii) fournir de nouvelles données sur la nature de l’interaction Hp/Ca2+ en utilisant une stratégie multi-approches combinant plusieurs techniques expérimentales et de calculs quantiques iii) étudier le processus CID de différents systèmes par des simulations de dynamique moléculaire. / Heparin (Hp) is a sulfated polysaccharide composed of repeating dissacharide units of hexauronic acid linked (α1→4) to an hexosamine residue that belongs to the family of glycosaminoglycans (GAGs). Sulfation can occur at the 6-O and/or N-positions of the gluscosamine, as well as the 2-O position of the hexauronic acid. GAGs usually exist as the O-linked side-chains of proteoglycans, associated with numerous important physiological activities, generally related to their interaction with diverse proteins. In some cases, this interaction can be influenced by the binding of natural metal ions to these Hp/protein complexes. Their role is usually pertained to the affinity, specificity and stability of these complexes. Despite its relevance, the mechanism by which the cation modulates heparin activity in Hp-protein complexes is largely unknown. A range of strategies and tools has been developed to facilitate the determination of primary structures of analyte molecules of interest via tandem mass spectrometry (MS/MS). In fact, structural characterization of heparin sulfated and its metal complexes has been sustained by the development of mass spectrometry techniques. In some cases, it was observed that upon collision-induced dissociation (CID) activation, some of these heparin-like isomers share nearly the same fragmentation patterns, turning the identification process into a complicated step. Nevertheless, a few years ago, our group showed that the gas-phase reactivity of metal ions can shed light into differentiating isomeric saccharides. These studies can be useful for two reasons: just for purely analytical purposes and also because the different behavior of the isomers towards the metal gives information about the Hp/Metal interaction. This might be important to explain the biological considerations mentioned before. Moreover, Dugourd et al recently reported the optical spectra and photodissociation patterns of different Hp oligosaccharides under UV irradiation. In terms of fragmentation pathways, it was observed that Ultraviolet photodissociation (UVPD) spectra appear to be more informative than CID due to additional cross-ring cleavages that provide information about the sulfate group location. Remarkably, the optical spectrum is characterized by an intense broad band centered at 240 nm for sulfated disaccharides. Following these findings, we became interested in coupling optical spectroscopy and theoretical calculations in heparin disaccharides and developing an alternative strategy to characterize these metal interactions. As described before, CID processes are commonly used in several fields and represent a very valuable tool in protein or carbohydrate characterization. Motivated by previous work published by W. Hase and K. Song, we collaborated with R.Spezia and T. Riera’s group in order to achieve a better understanding of the MS/MS process of protonated peptides and sugars. CID mechanisms of simple models, N-Formylalanylamide (HCO-Ala-NH2) and Galactose-6-Sulfate, were studied by QM+MM chemical dynamics simulations and MS/MS experiments.

none

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 148-155). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 148-155). Also available on the Internet.