Mass selective axial ejection of ions and mass analysis with a stability island with linear quadrupoles with added octopole fields are described. With mass selective axial ejection, quadrupoles with 2.0% and 2.6% added octopole fields have been tested and compared to a conventional quadrupole. The effects of trapping ions at different q values, excitation voltage, scan direction, balanced and unbalanced rf voltages on the rods, and dc applied between the rods have been investigated. The highest scan speeds and highest resolution are obtained with resonant excitation and ejection at high q (q = 0.8). With axial ejection, the quadrupole with a 2.0% added octopole field provides mass resolution and ejection efficiencies similar to a conventional rod set. Quadrupole, dipole and simultaneous dipole-dipole excitation between the x and y rod pairs were compared and no advantage was found with quadrupole or dipole-dipole excitation. The effects of scan speed were investigated and a resolution at half height of about 1600 is possible at scan speeds up to 5000 Th/s. Mass analysis using islands of stability was investigated with a quadrupole with2.0% added octopole field. The island of stability is formed with auxiliary excitation. The experiments confirm the predictions of the simulations. With the resolving dc applied to the quadrupole so that the Mathieu parameter a>0, conventional mass analysis with applied rf and dc and no auxiliary excitation is possible. In this case use of an island of stability yields similar peak shape and resolution. However with the polarity of the resolving dc reversed so that a<0, only very low resolution can be obtained; the added octopole prevents conventional mass analysis. By using a stability island when a<0, the resolution is substantially improved.

Portable mass spectrometers provide convenience for applications where conventional mass spectrometers are not suitable. However, a series of miniaturization issues show up in small mass spectrometers, specifically mass analyzers, that need to be thoroughly addressed before further miniaturization. The work in this dissertation focuses on miniaturization issues of ion trap mass analyzers. Space-charge is one of the major issues in small ion traps affecting their analytical performance. It limits ion trapping capacity when ion-ion repulsion causes spreading of a packet of ions. Simulation studies on the relationship between different trap dimensions and trapping capacity was done on a geometry-optimized cylindrical ion trap. A reasonable way of scaling the two important operating parameters (trapping voltage and trapping frequency as functions of the trap dimension) was discussed and applied in the simulation. The trapping capacity (N) decreased with the physical trap dimension (r0) as expected, and N is scaled exponentially as r0. Scaling laws for trapping parameters are proposed, confirmed by SIMION simulations that evaluate the space charge issue in small ion traps. This effect represents a practical limit in ion trap miniaturization.Geometry deviation is another issue that cannot be neglected in miniaturized ion traps, especially in small linear ion traps (LIT). The LIT our group is working on consists of an assembly of two plates, of which each was made by lithographically patterning a series of electrodes on an insulating plate. It is a promising way of expanding the trap capacity at a small trap dimension. However, misalignment of the two plates might seriously affect its performance, specifically resolution and signal intensity. Simulations were done on the misalignment of two-plate planar LIT in the six possible degrees of freedom (DOF) of misalignment between the two plates. Each DOF's influence on the mass resolution and the ion detection efficiency were discussed. Preliminary data from a previous ceramic plate design was collected while most of the misalignment experiments were done on an improved version. A platform was designed incorporating four motorized stages to precisely control the alignment of the ion trap in vacuum. The new plate design was demonstrated to achieve a better than unit resolution for toluene and deuterated toluene after the plates were aligned. The impact on the resolution and signal intensity from pitch, x-, y- and z-displacement were also experimentally studied.

I describe the miniaturization of a linear-type ion trap mass spectrometer for possible applications in portable chemical analysis, and demonstrate the advantages of using lithographically patterned electrode plates in realizing an ion trap with dimension r0 less than 1 mm. The focus of the work was to demonstrate the viability and feasibility of the patterned electrode approach to trap miniaturization, and also to discover potential obstacles to its use. Planar ceramic substrates were patterned with metal electrodes using photolithography. Plates that were originally used in a linear trap with a half-spacing (r0) of 2.19 mm were positioned much closer together such that r0 = 0.95 mm. A capacitive voltage divider provided different radiofrequency (RF) amplitudes to each electrode, and the capacitor values were adjusted to provide the correct electric field at this closer spacing. Electron ionization mass spectra of toluene and dichloromethane demonstrate instrument performance with better than unit mass resolution. Compared with the larger plate spacing, the signal intensity is reduced, corresponding to the reduced trapping capacity of the smaller device, but the mass resolution of the larger device is retained. A further miniaturized linear ion trap with a half-spacing of 362 µm was designed and tested. A series of obstacles and troubleshooting on ion source, analytical method, and electronics were present. These experiments show promise for further miniaturization using patterned ceramic plates and provide a guide for the ion trap miniaturization. The feasibility of a wire linear ion trap was also demonstrated. Unit mass resolution was obtained, indicating a promise for further optimization and miniaturization of the wire linear ion trap. In addition to the practical experiments on the miniaturized linear ion traps, I theoretically studied ion motion in the toroidal ion trap using SIMION simulations, which show classical chaotic behavior of single ions. The chaotic motion is a result of the non-linear components of the electric fields as established by the trap electrodes, and not by Coulombic interaction from other ions. The chaotic behavior was observed specifically in the ejection direction of ions located in non-linear resonance bands within and adjacent to the region of stable trapping. The non-linear bands crossing through the stability regions correspond to hexapole resonance conditions, while the chaotic ejection observed immediately adjacent to the stable trapping region corresponds to a "fuzzy" ejection boundary. Fractal-like patterns were obtained in a series of zoomed-in regions of the stability diagram.

Radio frequency (RF) ion traps based on the quadrupole device developed by Paul and Steinwedel utilize a dynamic electric field to spatially confine the trajectory of charged particles and may be employed as mass spectrometers by selectively ejecting trapped molecules based on the mass to charge ratio. Because of the inherent sensitivity and specificity of this process, ion trap mass spectrometers have become a popular scientific instrument. In the past two decades there has been a push to develop portable ion trap mass spectrometers for in situ mass analysis by geometrically scaling traps to smaller sizes. This decreases the power and vacuum requirements which allows field portable instruments to use smaller/less powerful vacuum pumps and batteries. This dissertation presents the process of miniaturizing the planar linear ion trap (PLIT) to a microscale radius in order to investigate the scaling limits of mass spectrometers. The ultimate end goal is the integration of a PLIT into a portable mass spectrometry system. The PLIT consists of two flat, non-conducting plates, on which fine metal electrodes are patterned using standard microfabrication processes, including photolithography. An RF field is distributed across the electrodes to create a quadrupole electromagnetic potential which traps ions based on their mass to charge ratio. While simple in concept, the PLIT has been developed over a ten-year period including an investigation of a variety of substrate materials and design geometries. This dissertation briefly reviews the optimal fabrication flow and why the stated parameters have advantages over other possible combinations in a coplanar ion trap. Since ion trap miniaturization reduces the trapping volume (which also worsens the SNR and resolution of a mass spectrum), a novel RF phase tracking circuit was developed to exploit a phase locked condition during double resonance ejection. This was implemented on the PLIT to increase SNR before constructing the µPLIT. Better than unit resolutions (0.5 Da, FWHM) and SNR improvements were observed.Lastly, the successful miniaturization of the PLIT to a microscale radius is presented. This was done by redesigning the electrodes on the PLIT surface to have an equivalent trap radius (ro) of 800 μm. The μPLIT successfully confined then resonantly ejected ions with resolutions of approximately 2-3 Da. The performance of the μPLIT was also tested over a range of pressures from 2.5-42×10-3 Torr and retained resolutions between 2.3-2.7 Da. Ultimately, the μPLIT was shown to retain resolutions viable for portable mass spectrometry at pressures in the tens of millitorr while consuming a factor of 3.38 less power than the unscaled PLIT.

Cytochrome P450 (CYP) family 4 constitutes monoxygenases responsible for hydroxylation of fatty acids and other lipids. For example, CYP4F3 metabolizes leukotrienes and CYP4F8 prostaglandin H. Importantly, six of the twelve CYP4 enzymes are orphans, i.e., with an unknown biological function. The catalytic activity of the enzyme CYP4F8 is known in seminal vesicles, but not in skin or psoriatic lesions, where CYP4F8 is highly expressed. The orphan CYP4F22 is also expressed in skin, and mutations in its gene has been linked to the rare skin disease lamellar ichthyosis, together with, inter alia, mutations in the genes of 12R-LOX and eLOX3. These enzymes appear to constitute a pathway producing hydroperoxides and epoxyalcohols from arachidonic acid. CYP4F22 is hypothesized to act in a consecutive step within this pathway. The aim of this thesis was to develop analytical methods to prepare and analyze hydroperoxides and epoxyalcohols derived from fatty acids by LC-MS/MS, and to investigate the catalytic performance of CYP4F8 and CYP4F22 for these substrates. The 12R-hydroperoxide of arachidonic acid (12R-HPETE) was prepared by autoxidation and separated from other hydroperoxides by chiral HPLC. MS/MS analysis showed that the hydroperoxides were unstable within the ion trap, but were stabilized by an increase in the isolation width. From the hydroperoxides, epoxyalcohols were generated by hematin treatment, and separated by normal phase HPLC. MS/MS spectra of several epoxyalcohols, derived both from arachidonic acid and linoleic acid, were characterized with aid of [2H]isotopomers and MS3 analysis. Apart from metabolic studies the thesis also include detailed information on MS/MS analysis of several oxygenated fatty acids, with proposed fragmentation mechanisms. The open reading frame of CYP4F22 was expressed in a recombinant yeast system, and LC-MS/MS analysis revealed that CYP4F22 catalyzed ω3 hydroxylation of arachidonic acid, but not any of the tested epoxyalcohols. In contrast, CYP4F8 metabolizes an epoxyalcohol derived from 12R-HPETE, 11R,12R-epoxy-10-hydroxyeicosatrienoic acid, to the ω3 hydroxy metabolite. Conclusively, it was demonstrated that LC-MS/MS could be used for the analysis and separation of hydroperoxides and epoxyalcohols for metabolic studies.

This dissertation presents advances in the development of planar electrode ion traps. An ion trap is a device that can be used in mass analysis applications. Electrode surfaces create an electric field profile that trap ionized molecules of an analyte. The electric fields can then be manipulated to mass-selectively eject ions out of the trap into a detector. The resulting data can be used to analyze molecular structure and composition of an unknown compound. Conventional ion traps require machined electrode surfaces to form the electric trapping field. This class of electrode presents significant obstacles when attempting to miniaturize ion traps to create portable mass spectrometers. Machined electrodes lose required precision in shape, smoothness, and alignment as trapping dimensions decrease. Simplified electrode geometries are essential to open the way to miniaturized ion traps. The planar electrode ion trap presents a simplified geometry that utilizes photolithography processes in its fabrication. Patterns of electrodes are patterned on a planar ceramic substrate. Electric fields generated by these patterns can be nearly identical to those of ideal ion traps. The microfabrication processes involve the challenge of patterning on ceramic, patterning on two sides of a substrate, and patterning on a substrate with high topographic features. Four successful designs of planar ion traps are presented in this work: the planar Paul, toroidal, coaxial, and linear ion trap. These four designs have different strengths and weaknesses. The planar Paul trap is simpler to design and operate, the toroidal has a larger ion storage volume and so can be a more sensitive instrument, and the coaxial trap is a hybrid planar Paul and toroidal trap. The linear trap combines the simplicity of the planar Paul trap with the increased storage capacity of the toroidal trap. This work presents how these four designs advance work in miniaturized ion traps. In addition, microfabrication techniques and trap performance for these designs are presented.

Ion traps used in mass spectrometers are of two classes. One class consists of traps having three electrode geometries which have rotational symmetry about central axis. They are called axially symmetric ion traps. Paul trap, Cylindrical Ion Trap(CIT) are examples in this class. Other class of traps contain 2D electric field inside them which has same profile along the central axis due to translational symmetry. Linear Ion Trap(LIT) and Rectilinear Ion Trap(RIT) are examples in this class. In the ideal hyperbolic geometries of Paul trap and LIT, electric field is a perfectly linear function of distance from the center of the trap. But when these ideal geometries are simplified in to simpler geometries of the CIT and the RIT for ease in machining, linearity of field, which is a specialty of Paul trap and LIT is lost. In this thesis, an effort is made to optimize the field within the traps by using split electrodes. The ring electrode of the CIT and both pairs of electrodes in the RIT are divided into more number of parts. Suitable voltages are applied on these parts to improve the linearity of the field. This thesis contains six chapters. Chapter 1 contains a background information about mass spectrometry. Chapter 2 discusses the Boundary Element Method (BEM) used to calculate charge distribution and Nelder-Mead method used for optimization. It also shows the calculation of multipoles. In Chapter 3, two new geometries namely split-electrode RIT and split-electrode CIT are considered with the objective of improving the linearity of electric field inside them. It is shown here that by applying certain external potential on various parts of split electrodes of these geometries, it is possible to improve the linearity of electric field inside them. In Chapter 4, capacitor models of new geometries proposed in chapter 3 are discussed. The use of external capacitors as a replacement to external power supply is also discussed in this chapter. InChapter5, study similar to that ofChapter3is carried out by splitting the geometries in more number of parts. The possibility of improved field profile is investigated by applying full potential to some of these parts and keeping other parts at ground potential. In Chapter 6, concluding remarks are discussed.

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Le long bio-polymère d'ADN est condensé à l’intérieur du noyau des cellules eukaryotes à l'aide de petites protéines appelées histones. En plus de leurs fonctions condensatrices,ces histones sont également la cible de nombreuses modifications post-traductionnelles(MPT), particulièrement au niveau de leur section N-terminale. Ces modifications réversibles font partie d’un code d’histones épi-génétique transmissible qui orchestre et module dynamiquement certains événements impliquant la chromatine, tels l’activation et la désactivation de gènes ainsi que la duplication et la réparation d’ADN. Ces modifications sont impliquées subséquemment dans la signalisation et la progression de cancers, tels que la leucémie. En conséquence, l'élucidation des modifications d’histones est importante pour comprendre leurs fonctions biologiques. Une méthodologie analytique a été mise au point en laboratoire pour isoler, détecter, et quantifier les MPT d’histones en utilisant une approche rapide à deux volets à l’aide d’outils bioinformatiques spécialisés. La méthodologie développée en laboratoire a été validée en utilisant des histones de souche sauvage ainsi que deux types d’histones mutants déficients en enzymes acétyltransferase. Des trois sources d’histones utilisées, la seule MPT qui a démontré un changement significatif est l’acétylation de l’histone H3 à lysine 56 (H3K56ac). L’expression et la stoechiométrie de cette MPT, issue de cellules de souche sauvage et de cellules mutantes, ont été déterminées avec précision et comparées. Les fonctions de balayage polyvalentes d'un instrument à trappe ionique quadrupôle linéaire hybride ont été utilisées pour améliorer la détection de protéines intactes. Le mode de balayage « enhanced multiply charged » (EMC) a été modifié pour contenir et détecter les ions de protéines intactes situées dans la trappe ionique linéaire. Ce mode de balayage nommé « targeted EMC » (tEMC) a permis de quadrupler le niveau de sensibilité (signal/interférence), et quintupler la résolution du mode de balayage conventionnel. De plus, la capacité de séparation des charges du tEMC a réduit de façon significative les effets de « space charge » dans la trappe ionique linéaire. La résolution supérieure du mode tEMC a permis de différencier plusieurs isoformes modifiées, particulièrement pour l’histone H3. L’analyse des peptides d’histones trypsiques à l’aide du mode de balayage « MRM » a permis le séquençage et la quantification de MPT avec un haut degré de précision. La seule MPT qui était sous-exprimée entre l’histone de souche sauvage et le mutant DOT1L fut la méthylation de l’histone H3 lysine 79(H3K79me1). Les effets de deux inhibiteurs d’enzymes HDAC (HDACi) sur l’expression de MPT d’histone ont été évalués en utilisant la méthodologie analytique mentionnée. Les histones extraites de cellules normales et cancéreuses ont été exposées à du Vorinostat(SAHA) ou du Entinostat (MS-275) pour une période de 24 à 72 heures. Deux histones furent principalement affectées, soit H3 et H4. Étonnamment, les mêmes effets n'ont pas été détectés lorsque les cellules normales ont été traitées avec le HDACi pour une période de 48 à 72 heures. Une méthode absolue de quantification avec une courbe d’étalonnage a été développée pour le peptide H3K56ac. Contrairement à certaines publications, nos résultats démontrent que cette MPT est présente dans les cellules mammifères avec une stoechiométrie très basse (< 0,1%) et n'est pas surexprimée de façon significative après le traitement au HDACi. / In eukaryotic cells, the lengthy DNA biopolymer is condensed into the cell nucleus with the aid of small packaging proteins called histones. In addition to their packing functions,histones are also targets of numerous post translational modifications (PTMs), especially on their N-terminus. These reversible modifications are believed to be constituents of a heritable epigenetic “histone code” that dynamically orchestrate and modulate chromatin based events such as gene activation and silencing, DNA replication and repair, and are also involved in the downstream signaling and progression of cancers, such as leukemia. Thus, the elucidation of histone PTMs is important in understanding their biological function. An analytical workflow was designed and set-up in the laboratory to isolate, detect, and quantitate histone PTM, using a two-pronged, unbiased, and rapid approach with specialized bioinformatic tools. The workflow was validated using histones from wildtype, and 2 mutants deficient in acetyltransferase activity. Between the three histone sources, the only PTM that demonstrated any change was acetylation at histone H3 lysine 56 (H3K56ac). The down-regulation and stoichiometry of this PTM was accurately assessed between wild-type and mutant cells. The versatile scan functions of a hybrid quadrupole-linear ion trap instrument were exploited to enhance the detection of intact histone proteins. The enhanced multiply charged (EMC) scan was modified in order to contain and detect intact protein ions within the linear ion trap. This targeted EMC (or tEMC) resulted in not only a 4-fold increase in signal-to-noise, but also a 5-fold increase in resolution. Furthermore, the charge separation capability of the tEMC dramatically reduced space charge effects within the linear ion trap. The superior resolution of the tEMC mode allowed for the discimination of many modified histone isoforms, especially for histone H3. Using the bottom-up strategy with multiple reaction monitoring (MRM), histone peptides were quantified and sequenced with a high degree of precision. The only PTM that was down-regulated between wild-type and DOT1L mutant histones was methylation at histone H3 lysine 79 (H3K79me1). The effects of two clinically relevant small molecule HDAC inhibitors (HDACi) on histone PTMs patterns were assessed using the analytical workflow developed. Histones derived from both normal and cancer cells were exposed to either Vorinostat (SAHA) or Entinostat (MS-275) over a 24- to 72 hour period. The two core histones primarily affected were H3 and H4. Surprisingly, the same effects were not observed when normal cells were treated with three doses of SAHA at 24-hour intervals over a 72-hour period. An absolute quantitation method using a calibration curve was developed for H3K56ac. In opposition to other published literature, our findings demonstrate that this PTM is present in very low stoichiometry (< 0.1%) in mammalian cells, and exhibits no significant up-regulation in different cell lines treated with several types of HDACi.

Le long bio-polymère d'ADN est condensé à l’intérieur du noyau des cellules eukaryotes à l'aide de petites protéines appelées histones. En plus de leurs fonctions condensatrices,ces histones sont également la cible de nombreuses modifications post-traductionnelles(MPT), particulièrement au niveau de leur section N-terminale. Ces modifications réversibles font partie d’un code d’histones épi-génétique transmissible qui orchestre et module dynamiquement certains événements impliquant la chromatine, tels l’activation et la désactivation de gènes ainsi que la duplication et la réparation d’ADN. Ces modifications sont impliquées subséquemment dans la signalisation et la progression de cancers, tels que la leucémie. En conséquence, l'élucidation des modifications d’histones est importante pour comprendre leurs fonctions biologiques. Une méthodologie analytique a été mise au point en laboratoire pour isoler, détecter, et quantifier les MPT d’histones en utilisant une approche rapide à deux volets à l’aide d’outils bioinformatiques spécialisés. La méthodologie développée en laboratoire a été validée en utilisant des histones de souche sauvage ainsi que deux types d’histones mutants déficients en enzymes acétyltransferase. Des trois sources d’histones utilisées, la seule MPT qui a démontré un changement significatif est l’acétylation de l’histone H3 à lysine 56 (H3K56ac). L’expression et la stoechiométrie de cette MPT, issue de cellules de souche sauvage et de cellules mutantes, ont été déterminées avec précision et comparées. Les fonctions de balayage polyvalentes d'un instrument à trappe ionique quadrupôle linéaire hybride ont été utilisées pour améliorer la détection de protéines intactes. Le mode de balayage « enhanced multiply charged » (EMC) a été modifié pour contenir et détecter les ions de protéines intactes situées dans la trappe ionique linéaire. Ce mode de balayage nommé « targeted EMC » (tEMC) a permis de quadrupler le niveau de sensibilité (signal/interférence), et quintupler la résolution du mode de balayage conventionnel. De plus, la capacité de séparation des charges du tEMC a réduit de façon significative les effets de « space charge » dans la trappe ionique linéaire. La résolution supérieure du mode tEMC a permis de différencier plusieurs isoformes modifiées, particulièrement pour l’histone H3. L’analyse des peptides d’histones trypsiques à l’aide du mode de balayage « MRM » a permis le séquençage et la quantification de MPT avec un haut degré de précision. La seule MPT qui était sous-exprimée entre l’histone de souche sauvage et le mutant DOT1L fut la méthylation de l’histone H3 lysine 79(H3K79me1). Les effets de deux inhibiteurs d’enzymes HDAC (HDACi) sur l’expression de MPT d’histone ont été évalués en utilisant la méthodologie analytique mentionnée. Les histones extraites de cellules normales et cancéreuses ont été exposées à du Vorinostat(SAHA) ou du Entinostat (MS-275) pour une période de 24 à 72 heures. Deux histones furent principalement affectées, soit H3 et H4. Étonnamment, les mêmes effets n'ont pas été détectés lorsque les cellules normales ont été traitées avec le HDACi pour une période de 48 à 72 heures. Une méthode absolue de quantification avec une courbe d’étalonnage a été développée pour le peptide H3K56ac. Contrairement à certaines publications, nos résultats démontrent que cette MPT est présente dans les cellules mammifères avec une stoechiométrie très basse (< 0,1%) et n'est pas surexprimée de façon significative après le traitement au HDACi. / In eukaryotic cells, the lengthy DNA biopolymer is condensed into the cell nucleus with the aid of small packaging proteins called histones. In addition to their packing functions,histones are also targets of numerous post translational modifications (PTMs), especially on their N-terminus. These reversible modifications are believed to be constituents of a heritable epigenetic “histone code” that dynamically orchestrate and modulate chromatin based events such as gene activation and silencing, DNA replication and repair, and are also involved in the downstream signaling and progression of cancers, such as leukemia. Thus, the elucidation of histone PTMs is important in understanding their biological function. An analytical workflow was designed and set-up in the laboratory to isolate, detect, and quantitate histone PTM, using a two-pronged, unbiased, and rapid approach with specialized bioinformatic tools. The workflow was validated using histones from wildtype, and 2 mutants deficient in acetyltransferase activity. Between the three histone sources, the only PTM that demonstrated any change was acetylation at histone H3 lysine 56 (H3K56ac). The down-regulation and stoichiometry of this PTM was accurately assessed between wild-type and mutant cells. The versatile scan functions of a hybrid quadrupole-linear ion trap instrument were exploited to enhance the detection of intact histone proteins. The enhanced multiply charged (EMC) scan was modified in order to contain and detect intact protein ions within the linear ion trap. This targeted EMC (or tEMC) resulted in not only a 4-fold increase in signal-to-noise, but also a 5-fold increase in resolution. Furthermore, the charge separation capability of the tEMC dramatically reduced space charge effects within the linear ion trap. The superior resolution of the tEMC mode allowed for the discimination of many modified histone isoforms, especially for histone H3. Using the bottom-up strategy with multiple reaction monitoring (MRM), histone peptides were quantified and sequenced with a high degree of precision. The only PTM that was down-regulated between wild-type and DOT1L mutant histones was methylation at histone H3 lysine 79 (H3K79me1). The effects of two clinically relevant small molecule HDAC inhibitors (HDACi) on histone PTMs patterns were assessed using the analytical workflow developed. Histones derived from both normal and cancer cells were exposed to either Vorinostat (SAHA) or Entinostat (MS-275) over a 24- to 72 hour period. The two core histones primarily affected were H3 and H4. Surprisingly, the same effects were not observed when normal cells were treated with three doses of SAHA at 24-hour intervals over a 72-hour period. An absolute quantitation method using a calibration curve was developed for H3K56ac. In opposition to other published literature, our findings demonstrate that this PTM is present in very low stoichiometry (< 0.1%) in mammalian cells, and exhibits no significant up-regulation in different cell lines treated with several types of HDACi.