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>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>My research has focused on the development and translation of ambient ionization mass spectrometry (MS)-based platforms in clinical and surgical settings, specifically in the area of brain cancer diagnostics and surgical decision making. Ambient ionization MS methods, such as those described herein, generate and analyze gas phase ions with high sensitivity and specificity from minimally prepared samples in near-real-time, on the order of seconds to minutes, rendering them well suited to point-of-care applications. We used ambient ionization MS methods, specifically desorption electrospray ionization mass spectrometry (DESI-MS) and extraction nanoelectrospray ionization mass spectrometry (nESI-MS) to molecularly characterize brain cancer biopsies. The characterization was made using diagnostic compounds identified as markers of disease state, tissue composition, tumor type, and genotype in human brain tissue. Methods were developed and validated offline in the laboratory and translated to clinical and surgical settings, thereby generating chemical information on prognostic features intraoperatively and providing valuable information that would be otherwise unavailable. We believe that, with approval, the methodologies described can assist physicians and improve patient outcomes by providing analytical tools and molecular information that can inform surgical decision making and adjuvant treatment strategies, complementing and not interfering with standard of care protocols.</p> <p><br></p> <p>We have successfully demonstrated the use of desorption electrospray ionization mass spectrometry (DESI-MS) for the expedient molecular assessment of human glioma tissue biopsies based on lipid profiles and prognostic metabolites, both at the tumor core and near surgical margins, in two small-scale, clinical studies. Maximal surgical resection of gliomas that avoids non-infiltrated tissue is associated with survival benefit in patients with glioma. The infiltrative nature of gliomas, as well as their morphological and genetic diversity, renders treatment difficult and demands an integrated imaging and diagnostic approach during surgery to guide clinicians in achieving maximal tumor resection. Further, the estimation of tumor cell percentage (TCP), a measure of tumor infiltration at surgical margins, is not routinely assessed intraoperatively. </p> <p>We have previously shown that rapid, offline molecular assessment of tumor infiltration in tissue biopsies is possible and believe that the same assessment performed intraoperatively in biopsied tissue near surgical margins could improve resection and better inform patient management strategies, including postoperative radiotherapy. Using a DESI-MS spectral library of normal brain tissue and glioma biopsies to generate a statistical model to classify brain tissue biopsies intraoperatively, multivariate statistical approaches were used to predict the disease state and tumor cell percentage (TCP) of each biopsy, thereby providing an measure of tumor infiltration at surgical margins via molecular indicators. In addition to assessment of tumor infiltration, we have developed DESI-MS assays for detecting the oncometabolite 2-Hydroxyglutarate (2HG) to detect isocitrate dehydrogenase (IDH) mutations in gliomas intraoperatively. Knowledge of IDH genotypes at the time of surgical resection could improve patient outcomes, as more aggressive tumor resection of IDH-mutated gliomas is associated with increased survival. While assessments of IDH genotype are typically not available until days after surgery, we have demonstrated the ability to provide this information is less than five minutes. An intraoperative DESI-MS system has successfully been used in a proof-of-concept clinical study and intraoperative performance validation of this platform is ongoing. The findings of these two studies as well as strengths, weaknesses, and areas of improvement for upcoming future iterations of the research are discussed.</p> <p><br></p> <p>Point-of-care applications necessitate the adaptation of MS methodologies to smaller devices. Miniature mass spectrometers (Mini MS) boast small footprints, simple operation, and low power consumption, noise levels, and cost, making them attractive candidates for point-of-care use. In a small-scale clinical study, we demonstrated the first application of a Mini MS for determination of IDH mutation status in gliomas intraoperatively. This study paves a path forward for the application of Mini MS in the OR. With its small footprint and low power consumption and noise level, this application of miniature mass spectrometers represents a simple and cost-effective platform for an important intraoperative measurement. </p> <p><br></p> <p>While MS-based methods of tissue analysis can detect molecular features of interest and rapidly produce large quantities of data, their inherent speed is rarely utilized because they are traditionally coupled with time-consuming separation techniques (e.g., chromatography). Ambient ionization MS, specifically DESI-MS, is well suited for high-throughput applications due to its lack of sample preparation and purification techniques. In an attempt to rapidly characterize microarrays of tissue biopsies, we developed a high-throughput DESI-MS (HT-DESI-MS) method for the rapid characterization of disease state, human brain tumor type, glioma classification, and detection of IDH mutations in tissue microarrays (TMA) of banked and fresh human brain tissue biopsies. We anticipate that HT-DESI-MS analysis of TMAs could become a standard tool for the generation of spectral libraries for sample classification, the identification of biomarkers through large-scale studies, the correlation of molecular features with anatomical features when coupled to digital pathology, and the assessment of drug efficacy. </p>