Nano-ESI mass spectrometry is an attractive analytical technique due to its high sensitivity and small sample consumption, which is especially important for research areas such as proteomics. However, current nano-ESI emitters become a bottleneck for nano-ESI to be widely applied because of problems such as clogging, poor robustness, large flow resistance, and poor spray efficiency for highly aqueous solutions. The objective of this thesis study is to address the problems associated with tapered emitters and provide alternative solutions by developing advanced nano-ESI emitters. Two strategies that were explored to improve the clogging resistance and robustness while maintaining comparable electrospray performances include the development of emitters with larger apertures and multiple channels. Following these strategies, five types of emitters were fabricated without tapering either internal or external diameters, which include a roughened open tubular emitter, a porous membrane-assisted emitter, a microstructured multiple channel photonic crystal fiber (MSF) emitter, a packed ODS bead emitter, and an entrapped ODS bead emitter. The successful transformation of MSF fibers to nanoelectrospray emitters demonstrates a new practical approach to expand the application of nano-ESI because of its availability, compatibility, precisely controlled channel dimensions, variety of channel patterns, and feasibility for surface modification. The fundamental mechanism of non-tapered emitters was studied at nano flow rates. The fact that a plume of mist, instead of single Taylor cone, is generated from multiple channel emitters at nano flow rates suggests multiple Taylor cones may be formed. The calculated sensitivity gains from a MSF emitter compared to a single-tip tapered emitter are related to the number of the orifices containing on a MSF emitter. The characterization of impacts of operational parameters on nanoelectrospray performances shows that non-tapered emitters are more robust and less dependent on the emitter’s fine positioning. It was also found that unlike tapered emitters, non-tapered emitters can be positioned ten times further from the orifice of a mass spectrometer, which is greatly beneficial to online sample manipulation and purification. Furthermore, the electrospray efficiency of spraying highly aqueous solutions (e.g. 90%) was greatly improved through the hydrophobic modification of a MSF emitter exit. / Thesis (Ph.D, Chemistry) -- Queen's University, 2008-09-17 19:07:12.847

Carotenoids are natural pigments that have antioxidation and antimutagenic abilities. They are produced with the help of new technological methods. For example, carotenoid yeast Rhodotorula glutinis produces -carotene with the yield of up to 6 – 10 mg/g of the dry substance. The method of the mass spectrometry with the nanoelectrospray in the positive mode was optimized for the determination of -carotene, lycopene and astaxanthin in this project. Ionizing voltage of 4 kV and the sample flow rate of 15 – 80 nl/min through the spray silica fused capillary with the internal diameter of 25 µm were found to be the optimum parameters of the analysis. A mixture of chloroform with the addition of ammonia was used as a spray solvent for both standard and cellular samples. During the process of ionization by nanoelectrospray, -carotene and lycopene form cation radical [M] • + with the molecular mass to charge ratio (m/z) of 536, while asthaxanthin forms the protonated molecule [M + H]+ with the m/z of 597. The partial lysis of individual Rhodotorula glutinis cells was demonstrated under microscope in the organic solvents tetrahydrofuran and dimethylsulfoxide. Chloroform, acetone, acetonitrille, methanol and isopropanol did not affect the cells after a 15 min treatment.

<p><b>In this thesis the fabrication and analytical evaluation of two new electrospray emitters utilized for mass spectrometry analysis is presented. The emitters are based on a new concept, where the spray orifice can be varied in size. The thesis is based on two papers.</b></p><p>All present-day nanoelectrospray emitters have fixed dimensions. The range of the applicable flow rate for such an emitter is therefore rather limited and exchange of emitters may be necessary from one experiment to another. Optimization of the signal of the analyte ions is also limited to adjustments of the applied voltage or the distance between the emitter and the mass spectrometer inlet. Furthermore, clogging can occur in emitters with fixed dimensions of narrow orifice sizes. In this thesis, electrospray emitters with a variable size of the spray orifice are proposed. An open gap between two thin substrates is filled with sample solution via a liquid bridge from a capillary. Electrospray is generated at the end point of the gap, which can be varied in width.</p><p>In Paper I, electrospray emitters fabricated in polyethylene terephthalate have been evaluated. Triangular tips are manually cut from the polymer film. The tips are mounted to form a gap between the edges of the tips. The gap wall surfaces are subjected to a hydrophilic surface treatment to increase the wetting of the gap walls.</p><p>In Paper II, silicon electrospray chips with high precision are fabricated and evaluated. A thin beam, elevated from the bulk silicon chip is fabricated by means of deep reactive ion etching. The top surfaces of the beams of two chips act as a sample conduit when mounted in the electrospray setup. An anisotropic etching step with KOH of the intersecting <100> crystal planes results in a very sharp spray point. The emitters were given a hydrophobic surface treatment except for the hydrophilic gap walls.</p><p>For both emitter designs, the gap width has been adjusted during the experiments without any interruption of the electrospray. For a continuously applied peptide mixture, a shift towards higher charge states and increased signal to noise ratios could be observed when decreasing the gap width. The limit of detection has been investigated and the silicon chips have been interfaced with capillary electrophoresis.</p>

In this thesis the fabrication and analytical evaluation of two new electrospray emitters utilized for mass spectrometry analysis is presented. The emitters are based on a new concept, where the spray orifice can be varied in size. The thesis is based on two papers. All present-day nanoelectrospray emitters have fixed dimensions. The range of the applicable flow rate for such an emitter is therefore rather limited and exchange of emitters may be necessary from one experiment to another. Optimization of the signal of the analyte ions is also limited to adjustments of the applied voltage or the distance between the emitter and the mass spectrometer inlet. Furthermore, clogging can occur in emitters with fixed dimensions of narrow orifice sizes. In this thesis, electrospray emitters with a variable size of the spray orifice are proposed. An open gap between two thin substrates is filled with sample solution via a liquid bridge from a capillary. Electrospray is generated at the end point of the gap, which can be varied in width. In Paper I, electrospray emitters fabricated in polyethylene terephthalate have been evaluated. Triangular tips are manually cut from the polymer film. The tips are mounted to form a gap between the edges of the tips. The gap wall surfaces are subjected to a hydrophilic surface treatment to increase the wetting of the gap walls. In Paper II, silicon electrospray chips with high precision are fabricated and evaluated. A thin beam, elevated from the bulk silicon chip is fabricated by means of deep reactive ion etching. The top surfaces of the beams of two chips act as a sample conduit when mounted in the electrospray setup. An anisotropic etching step with KOH of the intersecting &lt;100&gt; crystal planes results in a very sharp spray point. The emitters were given a hydrophobic surface treatment except for the hydrophilic gap walls. For both emitter designs, the gap width has been adjusted during the experiments without any interruption of the electrospray. For a continuously applied peptide mixture, a shift towards higher charge states and increased signal to noise ratios could be observed when decreasing the gap width. The limit of detection has been investigated and the silicon chips have been interfaced with capillary electrophoresis. / QC 20101108

No description available.

Proteins are a highly diversified group of molecules, andfor their study, advanced analytical tools are required. Inparticular, a need for high-throughput techniques has emergedin order to enable the characterization of large sets ofproteins. In this thesis, improved techniques for proteinseparations as well as new tools for the mass spectrometricanalysis of proteins are described. In the work, presented in the first part of the thesis, arefined extract containing proteases from Antarctic krill (Euphausia superba) was separated and characterized bymeans of capillary electrophoresis (CE) and mass spectrometry(MS). Tailored CE separations of the krill extract revealed thepresence of approximately 50 components. In addition, adetailed CE and MS analysis of fractions, containing individualkrill proteases has been carried out. Trypsin-like proteasesfrom krill exhibited a 12-fold and a 60-fold higher digestionefficiency at 37 °C and 2 °C respectively compared todigests performed with bovine trypsin. Furthermore, thecleavage specificity of the trypsin-like proteases wasstudied. In the last part of the thesis, novel concepts forchip-based nanoelectrospray (nanoESI) and matrix-assisted laserdesorption/ionization (MALDI) mass spectrometry are described.First, a micromachined silicon chip with a two-dimensionalmatrix of out-ofplane nanoESI needles for high-throughputanalysis was fabricated. A two-fold improvement insignal-to-noise reproducibility was obtained. Second, achip-based target for MALDI was developed, which featured pairsof elevated 50×50 µm anchors in close proximity. Theanchors were individually addressable with sample solution. Theminiaturized sample preparations at close distance to eachother allowed a simultaneous ionization of a physicallyseparated sample and standard by one single laser pulse. Thisresulted in a twofold reduction of relative mass errors.Moreover, ion suppression of the analyte was significantlyreduced. The effective utilization of the sample resulted in adetection limit of ca 200 zeptomole of angiotensin I. Key words:Proteins, peptides, proteases, Antarctickrill,Euphausia superba, capillary electrophoresis,fluorosurfactants, mass spectrometry, nanoelectrospray, ESI,MALDI, chip, high-throughput, reproducibility, sensitivity andmass accuracy.

In this thesis, new methods that address some current limitations in nanoelectrospray mass spectrometry (nESI-MS) analysis are presented. One of the major objectives is the potential gain in sensitivity that can be obtained when employing the proposed techniques. In the first part of this thesis, a new emitter, based on the generation of electrospray from a spray orifice with variable size, is presented. Electrospray is generated from an open gap between the edges of two individually mounted, pointed tips. The fabrication and evaluation of two different types of such emitters is presented; an ESI emitter fabricated from polyethylene terephtalate (Paper I), and a high-precision silicon device (Paper II). Both emitters were surface-treated in a selective way for an improved wetting of the gap and to confine the sample solution into the gap. In the second part of this thesis, different methods for improved sensitivity of nESI-MS analysis have been developed. In Paper III, a method for nESI-MS analysis from discrete sample volumes down to 1.5 nL is presented, using commercially available nESI needles. When analyzing attomole amounts of analyte in such a small volume of sample, an increased sensitivity was obtained, compared to when analyzing equal amounts in conventional nESI-MS analysis. To be able to analyze smaller sample volumes, needles with a narrower orifice and a higher flow resistance were needed. This triggered the development of a new method for fabrication of fused silica nESI needles (Paper IV). The fabrication is based on melting of a fused silica capillary by means of a rotating plasma, prior to pulling the capillary into a fine tip. Using the described technique, needles with sub-micrometer orifices could be fabricated. Such needles enabled the analysis of sample volumes down to 275 pL, and a further improvement of the sensitivity was obtained. In a final project (Paper V), nESI-MS was used to study the aggregation behavior of Aβ peptides, related to Alzheimer’s disease. An immunoprecipitation followed by nESI-MS was employed. This technique was also utilized to study the selectivity of the antibodies utilized. / QC 20101112

<p>Proteins are a highly diversified group of molecules, andfor their study, advanced analytical tools are required. Inparticular, a need for high-throughput techniques has emergedin order to enable the characterization of large sets ofproteins. In this thesis, improved techniques for proteinseparations as well as new tools for the mass spectrometricanalysis of proteins are described.</p><p>In the work, presented in the first part of the thesis, arefined extract containing proteases from Antarctic krill (<i>Euphausia superba</i>) was separated and characterized bymeans of capillary electrophoresis (CE) and mass spectrometry(MS). Tailored CE separations of the krill extract revealed thepresence of approximately 50 components. In addition, adetailed CE and MS analysis of fractions, containing individualkrill proteases has been carried out. Trypsin-like proteasesfrom krill exhibited a 12-fold and a 60-fold higher digestionefficiency at 37 °C and 2 °C respectively compared todigests performed with bovine trypsin. Furthermore, thecleavage specificity of the trypsin-like proteases wasstudied.</p><p>In the last part of the thesis, novel concepts forchip-based nanoelectrospray (nanoESI) and matrix-assisted laserdesorption/ionization (MALDI) mass spectrometry are described.First, a micromachined silicon chip with a two-dimensionalmatrix of out-ofplane nanoESI needles for high-throughputanalysis was fabricated. A two-fold improvement insignal-to-noise reproducibility was obtained. Second, achip-based target for MALDI was developed, which featured pairsof elevated 50×50 µm anchors in close proximity. Theanchors were individually addressable with sample solution. Theminiaturized sample preparations at close distance to eachother allowed a simultaneous ionization of a physicallyseparated sample and standard by one single laser pulse. Thisresulted in a twofold reduction of relative mass errors.Moreover, ion suppression of the analyte was significantlyreduced. The effective utilization of the sample resulted in adetection limit of ca 200 zeptomole of angiotensin I.</p><p><b>Key words:</b>Proteins, peptides, proteases, Antarctickrill,<i>Euphausia superba</i>, capillary electrophoresis,fluorosurfactants, mass spectrometry, nanoelectrospray, ESI,MALDI, chip, high-throughput, reproducibility, sensitivity andmass accuracy.</p>

<p>Mass spectrometry, the science and technology of ions, owes much of its current popularity to the development of electrospray ionization. The development of electrospray ionization, along with its low flow-rate analog nanoelectrospray ionization, has increased the chemical space that can be investigated using mass spectrometers by orders of magnitude. While the interfacial chemistry of charged microdroplets that are generated by nanoelectrospray has been studied in detail, the physics of their motion, particularly in the presence of an applied field at ambient pressures, remains relatively unexplored. In this dissertation, an increase in ion currents detected by a commercial triple quadrupole mass spectrometer is used to demonstrate that: (i) the orthogonal injection of counterions into an electrode assembly can compensate for space charge effects and enhance the sampling of charged microdroplets from a nanoelectrospray focused electrostatically under ambient conditions into the mass spectrometer; and (ii) the ease of ion evaporation from charged microdroplets may be elucidated for small molecules based on their relative transmission through an electrode assembly for the simultaneous ambient electrostatic focusing of two nanoelectrosprays. In each case, the development is characterized by using ion trajectory calculations in conjunction with experiments, using homebuilt devices designed and fabricated in-house as rapid prototypes via 3D printing. In the open air, charged microdroplets have low kinetic energies with a narrow energy spread. Despite these limitations, this dissertation demonstrates, through the electrostatic manipulation of charged microdroplets produced via nanoelectrospray ionization, that a better understanding of the physics of moving charges in the open air can be used to increase the sensitivity of atmospheric pressure ionization.</p>

<p>QC 20110615</p>