Matching Items (15)

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Optimization of a Blood-Based Tuberculosis Diagnostic Assay for the Developing World-with a focus on Sub-Saharan Africa

Description

The optimization of a blood-based assay for diagnosing tuberculosis which has been developed and validated in Dr. Hu’s lab, at Arizona State University, is important for ensuring its successful translation

The optimization of a blood-based assay for diagnosing tuberculosis which has been developed and validated in Dr. Hu’s lab, at Arizona State University, is important for ensuring its successful translation to a resource-limited setting of the developing world. Tuberculosis is most prevalent in the developing world with Sub-Saharan Africa having the highest cases of HIV/TB coinfections. The implementation of a blood-based assay for diagnosing Tuberculosis in the sub-Saharan would significantly improve the diagnosis and treatment monitoring of tuberculosis thereby managing or eliminating the pandemic altogether. The World Health Organization has called for robust diagnostic technologies that would resolve the shortfalls of the current technologies which include GeneXpert, X-ray, and smear microscopy. The blood-based diagnostic methodology heavily relies on Mass-spectrometry, a technology which could be entirely novel and expensive to implement in most laboratories in the Sub-Saharan. Despite virtual challenges in implementing the technology, the assay has demonstrated high specificity and sensitivity to HIV/TB coinfected patients and children in comparison to the available TB diagnostic assays. This study endorses the Blood-based Mass Spectrometry assay as one of the promising technologies to effectively improve the diagnosis of TB. The performance of the assay on detecting TB antigens was tested using different methods and materials. In the end, the use of DBS and miniaturized mass spectrometers have been discussed as possible routes for translating the assay to the developing world

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  • 2019-05

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Alternative Plate-based MSIA Protein Analysis Technique

Description

Biomarkers are the cornerstone of modern-day medicine. They are defined as any biological substance in or outside the body that gives insight to the body's condition. Doctors and researchers can

Biomarkers are the cornerstone of modern-day medicine. They are defined as any biological substance in or outside the body that gives insight to the body's condition. Doctors and researchers can measure specific biomarkers to diagnose and treat patients, such as the concentration of hemoglobin Alc and its connection to diabetes. There are a variety of methods, or assays, to detect biomarkers, but the most common assay is enzyme-linked immunosorbent assay (ELISA). A new-generation assay termed mass spectrometric immunoassay (MSIA) can measure proteoforms, the different chemical variations of proteins, and their relative abundance. ELISA on the other hand measures the overall concentration of protein in the sample. Measuring each of the proteoforms of a protein is important because only one or two variations could be biologically significant and/or cause diseases. However, running MSIA is expensive. For this reason, an alternative plate-based MSIA technique was tested for its ability to detect the proteoforms of a protein called apolipoprotein C-III (ApoC-III). This technique combines the protein capturing procedure of ELISA to isolate the protein with detection in a mass spectrometer. A larger amount of ApoC-III present in the body indicates a considerable risk for coronary heart disease. The precision of the assay is determined on the coefficient of variation (CV). A CV value is the ratio of standard deviation in relation to the mean, represented as a percentage. The smaller the percentage, the less variation the assay has, and therefore the more ability it has to detect subtle changes in the biomarker. An accepted CV would be less than 10% for single-day tests (intra-day) and less than 15% for multi-day tests (inter-day). The plate-based MSIA was started by first coating a 96-well round bottom plate with 2.5 micrograms of ApoC-III antibody. Next, a series of steps were conducted: a buffer wash, then the sample incubation, followed by another buffer wash and two consecutive water washes. After the final wash, the wells were filled with a MALDI matrix, then spotted onto a gold plate to dry. The dry gold target was then placed into a MALDI-TOF mass spectrometer to produce mass spectra for each spot. The mass spectra were calibrated and the area underneath each of the four peaks representing the ApoC-III proteoforms was exported as an Excel file. The intra-day CV values were found by dividing the standard deviation by the average relative abundance of each peak. After repeating the same procedure for three more days, the inter-day CVs were found using the same method. After completing the experiment, the CV values were all within the acceptable guidelines. Therefore, the plate-based MSIA is a viable alternative for finding proteoforms than the more expensive MSIA tips. To further validate this, additional tests will need to be conducted with different proteins and number of samples to determine assay flexibility.

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  • 2017-12

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The Formic Acid Cluster Distribution Observed with Femtosecond Laser Ionization

Description

Microsolvation studies have begun to shed the light on the impact that single water molecules have on the structure of a molecule. The difference in behavior that molecules show when

Microsolvation studies have begun to shed the light on the impact that single water molecules have on the structure of a molecule. The difference in behavior that molecules show when exposed to an increasing number of water molecules has been considered important but remains elusive. The cluster distributions of formic acid were studied for its known importance as an intermediate in the water gas shift reaction. Implementations of the water gas shift reaction range from a wide range of applications. Studies have proposed implementations such as variety such as making water on the manned mission to mars and as an industrial energy source. The reaction pathway of formic acid favors decarboxylation in solvated conditions but control over the pathway is an important field of study. Formic acid was introduced into a high vacuum system in the form of a cluster beam via supersonic expansion and was ionized with the second harmonic (400nm) of a pump-probe laser. Mass spectra showed a ‘magic’ 5,1 (formic acid, water) peak which showed higher intensity than was usually observed in clusters with 1 water molecule. Peak integration showed a higher relative abundance for the 5,1 cluster as well and showed the increased binding favorability of this conformation. As a result, there is an enhanced probability of molecules sticking together in this arrangement and this is due to the stable, cage-like structure that the formic acid forms when surrounding the water molecule.

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  • 2020-05

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Improving Peptide Identification in Shotgun Proteomics Using Deep Neural Networks

Description

In shotgun proteomics, liquid chromatography coupled to tandem mass spectrometry
(LC-MS/MS) is used to identify and quantify peptides and proteins. LC-MS/MS produces mass spectra, which must be searched by one

In shotgun proteomics, liquid chromatography coupled to tandem mass spectrometry
(LC-MS/MS) is used to identify and quantify peptides and proteins. LC-MS/MS produces mass spectra, which must be searched by one or more engines, which employ
algorithms to match spectra to theoretical spectra derived from a reference database.
These engines identify and characterize proteins and their component peptides. By
training a convolutional neural network on a dataset of over 6 million MS/MS spectra
derived from human proteins, we aim to create a tool that can quickly and effectively
identify spectra as peptides prior to database searching. This can significantly reduce search space and thus run time for database searches, thereby accelerating LCMS/MS-based proteomics data acquisition. Additionally, by training neural networks
on labels derived from the search results of three different database search engines, we
aim to examine and compare which features are best identified by individual search
engines, a neural network, or a combination of these.

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  • 2020-05

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Microanalysis for Oxygen Fugacity by Secondary Ion Mass Spectrometry

Description

Oxygen fugacity (ƒO2) is a thermodynamic variable used to represent the redox state of a material or a system. It is equivalent to the partial pressure of oxygen in a

Oxygen fugacity (ƒO2) is a thermodynamic variable used to represent the redox state of a material or a system. It is equivalent to the partial pressure of oxygen in a particular environment corrected for the non-ideal behavior of the gas. ƒO2 is often used to indicate the potential for iron to occur in a more oxidized or reduced state at a particular temperature and pressure in a natural system. Secondary ion mass spectrometry (SIMS) is a powerful analytical instrument that can be used to analyze elemental and isotopic compositional information about microscopic features within solid materials. SIMS analyses of the secondary ion energy distribution of semi-pure metals demonstrate that the energy spectrum of individual mass lines can provide information about alterations in its surface environment.

The application of high-resolution (see Appendix C) energy spectrum calibrations to natural ilmenite led to the investigation of zirconium (90Zr+) and niobium (93Nb+) as potential indicators of sample ƒO2. Energy spectrum measurements were performed on an array of ilmenite crystals from the earth’s upper mantle retrieved from kimberlites and from a reduced meteorite. In all studied materials, variability in the peak shape and width of the energy spectra has been correlated with inferred sample ƒO2. The best descriptor of this relationship is the full-width at half-maximum (FWHM; see Appendix C) of the energy spectra for each sample. It has been estimated that a 1eV change in the FWHM of 93Nb+ energy spectra is roughly equivalent to 1 log unit ƒO2. Simple estimates of precision suggest the FWHM values can be trusted to  1eV and sample ƒO2 can be predicted to ±1 log unit, assuming the temperature of formation is known.

The work of this thesis also explores the applicability of this technique beyond analysis of semi-pure metals and ilmenite crystals from kimberlites. This technique was applied to titanium oxides experimentally formed at known ƒO2 as well as an ilmenite crystal that showed compositional variations across the grain (i.e., core to rim chemical variations). Analyses of titanium oxides formed at known ƒO2 agree with the estimation that 1 eV change in the FWHM of 93Nb+ is equivalent to ~1 log unit ƒO2 (in all cases but one); this is also true for analyses of a natural ilmenite crystal with compositional variations across the grain.

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  • 2019

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Mass spectrometric and molecular analyses of biological agents in environmental compartments

Description

This thesis discusses the use of mass spectrometry and polymerase chain reaction (PCR), among other methods, to detect biomarkers of microorganisms in the environment. These methods can be used to

This thesis discusses the use of mass spectrometry and polymerase chain reaction (PCR), among other methods, to detect biomarkers of microorganisms in the environment. These methods can be used to detect bacteria involved in the degradation of environmental pollutants (bioremediation) or various single-celled pathogens, including those posing potential threats as bioterrorism agents. The first chapter introduces the hurdles in detecting in diverse environmental compartments in which they could be found, a select list of single-celled pathogens representing known or potential bioterrorism agents. These hurdles take the form of substances that interfere either directly or indirectly with the detection method. In the case of mass spectrometry-based detection, many of these substances (interferences) can be removed via effective sample pretreatment. Chapters 2 through 4 highlight specific methods developed to detect bioremediation or bioterrorism agents in environmental matrices. These methods are qualitative mass spectrometry, quantitative PCR, and quantitative mass spectrometry, respectively. The targeted organisms in these methods include several bioremediation agents, e.g. Pseudomonas putida F1 and Sphingomonas wittichii RW1, and bioterrorism agents, e.g. norovirus and Cryptosporidium parvum. In Chapter 2, I identify using qualitative mass spectrometry, biomarkers for three bacterial species involved in bioremediation. In Chapter 3, I report on a new quantitative PCR method suitable for monitoring of a key gene in yet another bioremediation agent, Sphingomonas wittichii RW1; furthermore, I apply this method to track the efficacy of bioremediation in bioaugmented environmental microcosms. In Chapter 4, I report on the development of new quantitative mass spectrometry methods for two organisms, S. wittichii RW1 and Cryptosporidium parvum, and evaluate two previously published methods for their applicability to the analysis of complex environmental samples. In Chapter 5, I review state-of-the-art methods for the detection of emerging biological contaminants, specifically viruses, in environmental samples. While this summary deals exclusively with viral pathogens, the advantages and remaining challenges identified are also applicable to all single-celled organisms in environmental settings. The suggestions I make at the end of this chapter are expected to be valid not only for future needs for emerging viruses but also for bacteria, eukaryotic pathogens, and prions. In general, it is advisable to continue the trend towards quantification and to standardize methods to facilitate comparison of results between studies.

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Date Created
  • 2012

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Statistical signal processing of ESI-TOF-MS for biomarker discovery

Description

Signal processing techniques have been used extensively in many engineering problems and in recent years its application has extended to non-traditional research fields such as biological systems. Many of these

Signal processing techniques have been used extensively in many engineering problems and in recent years its application has extended to non-traditional research fields such as biological systems. Many of these applications require extraction of a signal or parameter of interest from degraded measurements. One such application is mass spectrometry immunoassay (MSIA) which has been one of the primary methods of biomarker discovery techniques. MSIA analyzes protein molecules as potential biomarkers using time of flight mass spectrometry (TOF-MS). Peak detection in TOF-MS is important for biomarker analysis and many other MS related application. Though many peak detection algorithms exist, most of them are based on heuristics models. One of the ways of detecting signal peaks is by deploying stochastic models of the signal and noise observations. Likelihood ratio test (LRT) detector, based on the Neyman-Pearson (NP) lemma, is an uniformly most powerful test to decision making in the form of a hypothesis test. The primary goal of this dissertation is to develop signal and noise models for the electrospray ionization (ESI) TOF-MS data. A new method is proposed for developing the signal model by employing first principles calculations based on device physics and molecular properties. The noise model is developed by analyzing MS data from careful experiments in the ESI mass spectrometer. A non-flat baseline in MS data is common. The reasons behind the formation of this baseline has not been fully comprehended. A new signal model explaining the presence of baseline is proposed, though detailed experiments are needed to further substantiate the model assumptions. Signal detection schemes based on these signal and noise models are proposed. A maximum likelihood (ML) method is introduced for estimating the signal peak amplitudes. The performance of the detection methods and ML estimation are evaluated with Monte Carlo simulation which shows promising results. An application of these methods is proposed for fractional abundance calculation for biomarker analysis, which is mathematically robust and fundamentally different than the current algorithms. Biomarker panels for type 2 diabetes and cardiovascular disease are analyzed using existing MS analysis algorithms. Finally, a support vector machine based multi-classification algorithm is developed for evaluating the biomarkers' effectiveness in discriminating type 2 diabetes and cardiovascular diseases and is shown to perform better than a linear discriminant analysis based classifier.

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Date Created
  • 2012

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Hyphenation of a microfluidic platform with MALDI-TOF mass spectrometry for single cell analysis

Description

Cell heterogeneity is widely present in the biological world and exists even in an isogenic population. Resolving the protein heterogeneity at the single cell level is of enormous biological and

Cell heterogeneity is widely present in the biological world and exists even in an isogenic population. Resolving the protein heterogeneity at the single cell level is of enormous biological and clinical relevance. However, single cell protein analysis has proven to be challenging due to extremely low amount of protein in a single cell and the huge complexity of proteome. This requires appropriate sampling and sensitive detection techniques. Here, a new approach, microfluidics combined with MALDI-TOF mass spectrometry was brought forward, for the analysis of proteins in single cells. The detection sensitivity of peptides as low as 300 molecules and of proteins as low as 10^6 molecules has been demonstrated. Furthermore, an immunoassay was successfully integrated in the microfluidic device for capturing the proteins of interest and further identifying them by subsequent enzymatic digestion. Moreover, an improved microfluidic platform was designed with separate chambers and valves, allowing the absolute quantification by employing iTRAQ tags or an isotopically labeled peptide. The study was further extended to analyze a protein in MCF-7 cell lysate. The approach capable of identifying and quantifying protein molecules in MCF-7 cells is promising for future proteomic studies at the single cell level.

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Date Created
  • 2016

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To Be Or Not To B: Meteoritic Implications for the Galactic Environment of Solar System Formation

Description

Short-lived radionuclides (SLRs) once present in the solar nebula can be used to probe the Solar System’s galactic formation environment. Isotopic analyses reveal that the first solids formed in the

Short-lived radionuclides (SLRs) once present in the solar nebula can be used to probe the Solar System’s galactic formation environment. Isotopic analyses reveal that the first solids formed in the Solar System, calcium- and aluminum-rich inclusions (CAIs) in chondritic meteorites, formed with the live SLRs 10Be (t1/2 = 1.4 Ma) and 26Al (t1/2 = 0.7 Ma). Beryllium-10 is produced when high-energy ions, solar energetic particles or galactic cosmic rays (GCRs), spall nuclei in gas or dust. The most likely source of Solar System 10Be is inheritance of GCR-irradiated protosolar molecular cloud material, but only if all CAIs recorded the same initial 10Be abundance. The goal of this dissertation is to assess the homogeneity of 10Be by measuring CAIs for 10Be–10B isotope systematics, correlated to 26Al–26Mg and oxygen isotopes.

I synthesized appropriate standards for secondary ion mass spectrometry (SIMS) measurements of 10Be–10B, necessary for accurate determination of the 10Be/9Be ratio. I then analyzed 32 CAIs for 10Be–10B as well as 6 CAIs for 26Al–26Mg and 5 CAIs for oxygen isotopes within this sample set using SIMS. Previous studies analyzed CAIs primarily from CV3 chondrites, which are known to have experienced thermal metamorphism and aqueous alteration. My work included a variety of CAIs (Type A, B, fine-grained, igneous) from CV3oxidized, CV3reduced, CO3, CR2, and CH/CB chondrites. Finally, after evaluating my data and literature data consistently, I statistically tested whether all CAIs belong to a single 10Be population. I find that the majority (~85%) of the normal (i.e., without large isotopic fractionations or anomalies), 26Al-bearing CAIs recorded a single value, 10Be/9Be = (7.0 ± 0.2) × 10-4. Although 6 CAIs recorded higher or lower values, these are plausibly explained by secondary alteration processes. The galaxy-wide average value of 10Be/9Be from GCR interactions 4.56 billion years ago is predicted to be <2 × 10-4; the value I measured is more than 3 times higher. Because GCRs trace supernovae and star formation, my results suggest a similarly enhanced star formation rate in the molecular cloud within ~1 kpc of the Sun, in the ~15 Ma prior to the Sun’s birth.

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  • 2020

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High resolution spectroscopy of metal-containing molecules and construction of resonance-enhanced multi-photon ionization time-of-flight mass spectrometer (REMPI-TOFMS)

Description

This thesis describes the studies for two groups of molecules in the gas-phase: (a) copper monofluoride (CuF) and copper hydroxide (CuOH); (b) thorium monoxide (ThO) and tungsten carbide (WC). Copper-containing

This thesis describes the studies for two groups of molecules in the gas-phase: (a) copper monofluoride (CuF) and copper hydroxide (CuOH); (b) thorium monoxide (ThO) and tungsten carbide (WC). Copper-containing molecules (Group a) are selected to investigate the ionic bonding in transition metal-containing molecules because they have a relatively simple electronic state distribution due to the nearly filled 3d-orbital. ThO and WC (Group b) are in support of particle physics for the determination of electron electric dipole moment (eEDM), de, the existence of which indicates new physics beyond the Standard Model. The determination of the tiny eEDM requires large electric fields applied to the electron. The 3(Delta)1 states for heavy polar molecules were proposed [E. R. Meyer, J. L. Bohn, and M. P. Deskevich, Phys. Rev. A 73, 062108 (2006)] to determine de with the following attractive features: (1) large electric dipole moments; (2) large internal electric fields, Eeff, experienced by valence electrons; (3) nearly degenerate omega-doublets; (4) extremely small magnetic dipole moments. The H3(Delta)1 state for ThO and the X3(Delta)1 state for WC are both good candidates. Spectroscopic parameters (i.e. molecular electric and magnetic dipole moments, omega-doubling parameters, etc) are required for the 3(Delta)1 states of ThO and WC. High resolution optical spectra (linewidth ~50 MHz) of CuF, CuOH, ThO and WC were recorded field-free and in the presence of a static electric field (or magnetic field) using laser ablation source/supersonic expansion and laser induced fluorescence (LIF) detection. The spectra were modeled by a zero-field effective Hamiltonian operator and a Stark (or Zeeman) Hamiltonian operator with various molecular parameters. The determined molecular parameters are compared to theoretical predictions. The small omega-doubling parameter was well determined using the pump/probe microwave optical double resonance (PPMODR) technique with a much higher resolution (linewidth ~60 kHz) than optical spectroscopy. In addition to the above mentioned studies of the two groups of molecules, a resonance enhanced multi-photon ionization (REMPI) combined with a time-of-flight mass spectrometer (TOFMS) has been developed to identify the molecules responsible for observed LIF signals. The operation of this spectrometer has been tested by recording the mass spectrum of Ti/O2 and the REMPI spectrum for TiO using a two-color excitation scheme.

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Date Created
  • 2012