Matching Items (23)
Filtering by
- All Subjects: Biochemistry
- Creators: Borges, Chad
Description
Cancer claims hundreds of thousands of lives every year in US alone. Finding ways for early detection of cancer onset is crucial for better management and treatment of cancer. Thus, biomarkers especially protein biomarkers, being the functional units which reflect dynamic physiological changes, need to be discovered. Though important, there are only a few approved protein cancer biomarkers till date. To accelerate this process, fast, comprehensive and affordable assays are required which can be applied to large population studies. For this, these assays should be able to comprehensively characterize and explore the molecular diversity of nominally "single" proteins across populations. This information is usually unavailable with commonly used immunoassays such as ELISA (enzyme linked immunosorbent assay) which either ignore protein microheterogeneity, or are confounded by it. To this end, mass spectrometric immuno assays (MSIA) for three different human plasma proteins have been developed. These proteins viz. IGF-1, hemopexin and tetranectin have been found in reported literature to show correlations with many diseases along with several carcinomas. Developed assays were used to extract entire proteins from plasma samples and subsequently analyzed on mass spectrometric platforms. Matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometric techniques where used due to their availability and suitability for the analysis. This resulted in visibility of different structural forms of these proteins showing their structural micro-heterogeneity which is invisible to commonly used immunoassays. These assays are fast, comprehensive and can be applied in large sample studies to analyze proteins for biomarker discovery.
ContributorsRai, Samita (Author) / Nelson, Randall (Thesis advisor) / Hayes, Mark (Thesis advisor) / Borges, Chad (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2012
Description
Protein AMPylation is a recently discovered and relatively unstudied post-translational modification (PTM). AMPylation has previously been shown to play an important role in metabolic regulation and host pathogenesis in bacteria, but the recent identification of potential AMPylators across many species in every domain of life has supported the possibility that AMPylation could be a more fundamental and physiologically significant regulatory PTM. For the first time, we characterized the auto-AMPylation capability of the human protein SOS1 through in vitro AMPylation experiments using full-length protein and whole-domain truncation mutants. We found that SOS1 can become AMPylated at a tyrosine residue possibly within the Cdc25 domain of the protein, the Dbl homology domain is vital for efficient auto-AMPylation activity, and the C-terminal proline-rich domain exhibits a complex regulatory function. The proline-rich domain alone also appears to be capable of catalyzing a separate, unidentified covalent self-modification using a fluorescent ATP analogue. Finally, SOS1 was shown to be capable of catalyzing the AMPylation of two endogenous human protein substrates: a ubiquitous, unidentified protein of ~49kDa and another breast-cancer specific, unidentified protein of ~28kDa.
ContributorsOber-Reynolds, Benjamin John (Author) / LaBaer, Joshua (Thesis director) / Borges, Chad (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
In this thesis, glycan nodes, the basic subunits of complex biological sugars, were studied to determine the reproducibility of gas chromatography-mass spectrometry (GC/MS) based methylation analysis of whole blood plasma by normalization using an internal standard of heavy permethylated glycans. Glycans are complex biological sugars that have a variety of applications in the human body and will display aberrant compositions when produced by cancerous cells. Thus an assay to determine their composition can be used as a diagnostic tool. It was shown that the assay may have potential use, but needs further refinement to become an improvement over current methods by analyzing the results of ratio-determination and replicate experiments.
ContributorsMiyasaki, Tyler Takeo (Author) / Borges, Chad (Thesis director) / Van Horn, Wade (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Chemical Engineering Program (Contributor)
Created2015-05
Description
Dielectrophoresis is a separations strategy that has the potential to separate small amounts of different proteins from each other. The forces at play in the channel used for dielectrophoresis are electroosmotic flow (EOF), electrophoresis (EP), and dielectrophoresis (DEP). EOF is the force exerted on liquid from an applied potential (1). EP is the force exerted on charged particles in a uniform electric field (2). DEP is the force exerted on particles (charged and uncharged) in a non-uniform electric field (3). This experiment was focused on the testing of a new microfluidic device to see if it could improve the focusing of proteins in dielectrophoresis. It was predicted that the addition of a salt bridge would improve focusing by preventing the ions created by the electrolysis of water around the electrodes from interacting with the proteins and causing aggregation, among other problems. Control trials using the old device showed that electrolysis was likely occurring and was the causal agent for poor outcomes. After applying the electric potential for some time a pH front traveled through the channel causing aggregation of proteins and the current in the channel decreased rapidly, even while the voltage was held constant. The resistance in the channels of the control trials also slightly decreased over time, until the pH shift occurred, at which time it increased rapidly. Experimental trials with a new device that included salt bridges eliminated this pH front and had a roughly linear increase of current in the channel with the voltage applied. This device can now be used in future research with protein dielectrophoresis, including in the potential differentiation of different proteins. References: 1) Electroosmosis. Oxford Dictionary of Biochemistry and Molecular Biology. 2. Oxford University Press: Oxford, England. 2006. 2) Electrophoresis. Oxford Dictionary of Biochemistry and Molecular Biology. 2. Oxford University Press: Oxford, England. 2006. 3) Dielectrophoresis. Oxford Dictionary of Biochemistry and Molecular Biology. 2. Oxford University Press: Oxford, England. 2006.
ContributorsHayes, Katelyn Donna (Author) / Hayes, Mark (Thesis director) / Borges, Chad (Committee member) / School of Life Sciences (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Currently, quantification of single cell RNA species in their natural contexts is restricted due to the little number of parallel analysis. Through this, we identify a method to increase the multiplexing capacity of RNA analysis for single cells in situ. Initially, RNA transcripts are found by using fluorescence in situ hybridization (FISH). Once imaging and data storage is completed, the fluorescence signal is detached through photobleaching. By doing so, the FISH is reinitiated to detect other RNA species residing in the same cell. After reiterative cycles of hybridization, imaging and photobleaching, the identities, positions and copy numbers of a huge amount of varied RNA species can be computed in individual cells in situ. Through this approach, we have evaluated seven different transcripts in single HeLa cells with five reiterative RNA FISH cycles. This method has the ability to detect over 100 varied RNA species in single cells in situ, which can be further applied in studies of systems biology, molecular diagnosis and targeted therapies.
ContributorsJavangula, Saiswathi (Author) / Guo, Jia (Thesis director) / Liang, Jianming (Committee member) / School of Molecular Sciences (Contributor) / School of Nutrition and Health Promotion (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Plasma and serum are the most commonly used liquid biospecimens in biomarker research. These samples may be subjected to several pre-analytical variables (PAVs) during collection, processing and storage. Exposure to thawed conditions (temperatures above -30 °C) is a PAV that is hard to control, and track and could provide misleading information, that fail to accurately reveal the in vivo biological reality, when unaccounted for. Hence, assays that can empirically check the integrity of plasma and serum samples are crucial. As a solution to this issue, an assay titled ΔS-Cys-Albumin was developed and validated. The reference range of ΔS-Cys-Albumin in cardio vascular patients was determined and the change in ΔS-Cys-Albumin values in different samples over time course incubations at room temperature, 4 °C and -20 °C were evaluated. In blind challenges, this assay proved to be successful in identifying improperly stored samples individually and as groups. Then, the correlation between the instability of several clinically important proteins in plasma from healthy and cancer patients at room temperature, 4 °C and -20 °C was assessed. Results showed a linear inverse relationship between the percentage of proteins destabilized and ΔS-Cys-Albumin regardless of the specific time or temperature of exposure, proving ΔS-Cys-Albumin as an effective surrogate marker to track the stability of clinically relevant analytes in plasma. The stability of oxidized LDL in serum at different temperatures was assessed in serum samples and it stayed stable at all temperatures evaluated. The ΔS-Cys-Albumin requires the use of an LC-ESI-MS instrument which limits its availability to most clinical research laboratories. To overcome this hurdle, an absorbance-based assay that can be measured using a plate reader was developed as an alternative to the ΔS-Cys-Albumin assay. Assay development and analytical validation procedures are reported herein. After that, the range of absorbance in plasma and serum from control and cancer patients were determined and the change in absorbance over a time course incubation at room temperature, 4 °C and -20 °C was assessed. The results showed that the absorbance assay would act as a good alternative to the ΔS-Cys-Albumin assay.
ContributorsJehanathan, Nilojan (Author) / Borges, Chad (Thesis advisor) / Guo, Jia (Committee member) / Van Horn, Wade (Committee member) / Arizona State University (Publisher)
Created2022
Description
Based on past studies, urinary glycan biomarkers have the potential to be used as diagnostic and prognostic markers for treatment purposes. This study brought into play the bottom-up glycan node analysis approach to analyze 39 urine samples from COVID-19 positive and negative individuals using gas chromatography-mass spectrometry (GC-MS) to determine potential urinary glycan biomarkers of COVID-19. Glycan node analysis involves chemically breaking down glycans in whole biospecimens in a way that conserves both monosaccharide identity and linkage information that facilitates the capture of unique glycan features as single analytical signals. Following data acquisition, the student t-test was done on all the nodes, but only four prominent nodes (t-Deoxyhexopyranose, 2,3-Gal, t-GlcNAc, and 3,6-GalNAc with respective p-values 0.03027, 0.03973, 0.0224, and 0.0004) were below the threshold p-value of 0.05 and showed some differences in the mean between both groups. To eliminate the probability of having false positive p-values, Bonferroni correction was done on the four nodes but only the 3,6-GalNAc node emerged as the only node that was below the newly adjusted p-value. Because sample analyses were done in batches, the Kruskal Wallis test was done to know if the batch effect was responsible for the observed lower relative concentration of 3,6-GalNAc in COVID-19 positive patients than in negative patients. A receiver operating characteristic curve (ROC) was plotted for the 3,6-GalNAc node and the area under the curve (AUC) was calculated to be 0.84, casting the 3,6-GalNAc node was a potential biomarker of COVID-19. 3,6-GalNAc largely arises from branched O-glycan core structures, which are abundant in mucin glycoproteins that line the urogenital tract. Lowered relative concentrations of 3,6-GalNAc in the urine of COVID-19 positive patients may be explained by compromised kidney function that allows non-mucinous glycoproteins from the blood to contribute a greater proportion of the relative glycan node signals than in COVID-19 negative patients. Future prospective clinical studies will be needed to validate both the biomarker findings and this hypothesis.
ContributorsEyonghebi Tanyi, Agbor (Author) / Borges, Chad R (Thesis advisor) / Mills, Jeremy H (Committee member) / Guo, Jia (Committee member) / Arizona State University (Publisher)
Created2023
Description
There are limited methods and techniques to quantitatively assess protein content in single cells or small cell populations of tissues. The standard protein insulin was used to understand how potential changes in the preparation or co-crystallization process could improve sensitivity and limit of detection through matrix assisted laser desorption ionization (MALDI) mass spectrometry analysis in Bruker’s Microflex LRF using polydimethylsiloxane (PDMS) reservoirs. In addition, initial imaging tests were performed on Bruker’s RapifleX MALDI Tissuetyper to determine the instrument’s imaging capabilities on proteins of interest through the use of a single layer “Christmas tree” microfluidic device, with the aim of applying a similar approach to future tissue samples. Data on 2µM insulin determined that a 95% laser power in the Microflex corresponded to 12-15% laser power in the RapifleX. Based on the experiments with insulin, the process of mixing insulin and saturated ɑ-Cyano-4-hydroxycinnamic acid (HCCA) matrix solvent in a 1:1 ratio using 10mM sodium phosphate buffer under area analysis is most optimized with a limit of detection value of 110 nM. With this information, the future aim is to apply this method to a double layer Christmas tree device in order to hopefully quantitatively analyze and image protein content in single or small cell populations.
ContributorsKow, Keegan (Author) / Ros, Alexandra (Thesis director) / Borges, Chad (Committee member) / Cruz-Villarreal, Jorvani (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor)
Created2023-05
Description
Transient protein-protein and protein-molecule interactions fluctuate between associated and dissociated states. They are widespread in nature and mediate most biological processes. These interactions are complex and are strongly influenced by factors such as concentration, structure, and environment. Understanding and utilizing these types of interactions is useful from both a fundamental and design perspective. In this dissertation, transient protein interactions are used as the sensing element of a biosensor for small molecule detection. This is done by using a transcription factor-small molecule pair that mediates the activation of a CRISPR/Cas12a complex. Activation of the Cas12a enzyme results in an amplified readout mechanism that is either fluorescence or paper based. This biosensor can successfully detect 9 different small molecules including antibiotics with a tuneable detection limit ranging from low µM to low nM. By combining protein and nucleic acid-based systems, this biosensor has the potential to report on almost any protein-molecule interaction, linking this to the intrinsic amplification that is possible when working with nucleic acid-based technologies. The second part of this dissertation focuses on understanding protein-molecule interactions at a more fundamental level, and, in so doing, exploring design rules required to generalize sensors like the ones described above. This is done by training a neural network algorithm with binding data from high density peptide micro arrays incubated with specific protein targets. Because the peptide sequences were chosen simply to evenly, though sparsely, represent all sequence space, the resulting network provides a comprehensive sequence/binding relationship for a given target protein. While past work had shown that this works well on the arrays, here I have explored how well the neural networks thus trained, predict sequence-dependent binding in the context of protein-protein and peptide-protein interactions. Amino acid sequences, either free in solution or embedded in protein structure, will display somewhat different binding properties than sequences affixed to the surface of a high-density array. However, the neural network trained on array sequences was able to both identify binding regions in between proteins and predict surface plasmon resonance-based binding propensities for peptides with statistically significant levels of accuracy.
ContributorsSwingle, Kirstie Lynn (Author) / Woodbury, Neal W (Thesis advisor) / Green, Alexander A (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2022
Purification, Characterization, and Structural Determination of Proteins Vital to Infectious Disease
Description
The work in this dissertation progressed the research of structural discovery for two targets critical in the fight of infectious disease. Francisella lipoprotein 3 (Flpp3) is a virulent determinant of tularemia and was the first protein of study. The proteins soluble domain was studied using a hybrid modeling theory that used small angle X-ray scattering (SAXS) in combination with computation analysis to generate a SAXS-refined structure. The SAXS-refined structure closely resembled the NMR structure (PDB: 2MU4) which contains a hydrophobic cavity inside the protein that could be used for drug discovery purposes. The full-length domain of Flpp3 purified from the outer membrane of E. coli was also studied with a combination of biophysical characterization methods. Mass spectrometry and western blot analysis confirmed Flpp3 being translocated to the outer membrane, while SDS-PAGE confirmed the purity of Flpp3 in the monomeric form after size exclusion chromatography. Using Circular Dichroism (CD) the monomeric form of Flpp3 was shown to be almost fully refolded into having a primarily β-stranded secondary structure. This information advances the progress of both tularemia research and outer membrane protein research as no natively folded outer membrane protein structures have been solved for F. tularensis.The second protein worked on in this dissertation is the nonstructural protein 15 from SARS-CoV-2, also called NendoU. Nsp15 is an endoribonuclease associated with aiding the virus responsible for the current COVID-19 pandemic in evasion of the immune system. An inactive mutant of Nsp15 was studied with both negative stain electron microscopy and cryogenic electron microscopy (Cryo-EM) in the presence of RNA or without RNA present. The initial findings of negative stain electron microscopy of Nsp15 with and without RNA showed a difference in appearance. Negative stain analysis of Nsp15 is in the presence of a 5nt RNA sequence in low salt conditions shows a conformational change when compared to Nsp15 without RNA present. As well the presence of RNA appeared to shift the electron density in Cryo-EM studies of Nsp15. This work advances the research in how Nsp15 may bind and cleave RNA and aid in the evasion of the host cell immune system.
ContributorsGoode, Matthew (Author) / Fromme, Petra (Thesis advisor) / Guo, Jia (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2022