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Description
The need for a renewable and sustainable light-driven energy source is the motivation for this work, which utilizes a challenging, yet practical and attainable bio-inspired approach to develop an artificial oxygen evolving complex, which builds upon the principles of the natural water splitting mechanism in oxygenic photosynthesis. In this work,

The need for a renewable and sustainable light-driven energy source is the motivation for this work, which utilizes a challenging, yet practical and attainable bio-inspired approach to develop an artificial oxygen evolving complex, which builds upon the principles of the natural water splitting mechanism in oxygenic photosynthesis. In this work, a stable framework consisting of a three-dimensional DNA tetrahedron has been used for the design of a bio-mimic of the Oxygen-Evolving Complex (OEC) found in natural Photosystem II (PSII). PSII is a large protein complex that evolves all the oxygen in the atmosphere, but it cannot be used directly in artificial systems, as the light reactions lead to damage of one of Photosystem II's core proteins, D1, which has to be replaced every half hour in the presence of sunlight. The final goal of the project aims to build the catalytic center of the OEC, including the Mn4CaCl metal cluster and its protein environment in the stable DNA framework of a tetrahedron, which can subsequently be connected to a photo-stable artificial reaction center that performs light-induced charge separation. Regions of the peptide sequences containing Mn4CaCl ligation sites are implemented in the design of the aOEC (artificial oxygen-evolving complex) and are attached to sites within the tetrahedron to facilitate assembly. Crystals of the tetrahedron have been obtained, and X-ray crystallography has been used for characterization. As a proof of concept, metal-binding peptides have been coupled to the DNA tetrahedron which allowed metal-containing porphyrins, specifically Fe(III) meso-Tetra(4-sulfonatophenyl) porphyrin chloride, to be encapsulated inside the DNA-tetrahedron. The porphyrins were successfully assembled inside the tetrahedron through coordination of two terminal histidines from the orthogonally oriented peptides covalently attached to the DNA. The assembly has been characterized using Electron Paramagnetic Resonance (EPR), optical spectroscopy, Dynamic Light Scattering (DLS), and x-ray crystallography. The results reveal that the spin state of the metal, iron (III), switches during assembly from the high-spin state to low-spin state.
ContributorsRendek, Kimberly Nicole (Author) / Fromme, Petra (Thesis advisor) / Chen, Julian (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2012
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Description
Spider dragline silk is well known for its outstanding mechanical properties - a combination of strength and extensibility that makes it one of the toughest materials known. Two proteins, major ampullate spidroin 1 (MaSp1) and 2 (MaSp2), comprise dragline silk fibers. There has been considerable focus placed on understanding the

Spider dragline silk is well known for its outstanding mechanical properties - a combination of strength and extensibility that makes it one of the toughest materials known. Two proteins, major ampullate spidroin 1 (MaSp1) and 2 (MaSp2), comprise dragline silk fibers. There has been considerable focus placed on understanding the source of spider silk's unique mechanical properties by investigating the protein composition, molecular structure and dynamics. Chemical compositional heterogeneity of spider silk fiber is critical to understand as it provides important information for the interactions between MaSp1 and MaSp2. Here, the amino acid composition of dragline silk protein was precisely determined using a solution-state nuclear magnetic resonance (NMR) approach on hydrolyzed silk fibers. In a similar fashion, solution-state NMR was applied to probe the "13"C/"15"N incorporation in silk, which is essential to understand for designing particular solid-state NMR methods for silk structural characterization. Solid-state NMR was used to elucidate silk protein molecular dynamics and the supercontraction mechanism. A "2"H-"13"C heteronuclear correlation (HETCOR) solid-state NMR technique was developed to extract site-specific "2"H quadrupole patterns and spin-lattice relaxation rates for understanding backbone and side-chain dynamics. Using this technique, molecular dynamics were determined for a number of repetitive motifs in silk proteins - Ala residing nanocrystalline &beta-sheet; domains, 3"1"-helical regions, and, Gly-Pro-Gly-XX &beta-turn; motifs. The protein backbone and side-chain dynamics of silk fibers in both dry and wet states reveal the impact of water on motifs with different secondary structures. Spider venom is comprised of a diverse range of molecules including salts, small organics, acylpolyamines, peptides and proteins. Neurotoxins are an important family of peptides in spider venom and have been shown to target and modulate various ion channels. The neurotoxins are Cys-rich and share an inhibitor Cys knot (ICK) fold. Here, the molecular structure of one G. rosea tarantula neurotoxin, GsAF2, was determined by solution-state NMR. In addition, the interaction between neurotoxins and model lipid bilayers was probed with solid-state NMR and negative-staining (NS) transmission electron microscopy (TEM). It is shown that the neurotoxins influence lipid bilayer assembly and morphology with the formation of nanodiscs, worm-like micelles and small vesicles.
ContributorsShi, Xiangyan (Author) / Yarger, Jeffery L (Thesis advisor) / Holland, Gregory P (Thesis advisor) / Levitus, Marcia (Committee member) / Marzke, Robert F (Committee member) / Arizona State University (Publisher)
Created2014
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Description
Rapid and reliable separation and analysis of proteins require powerful analytical methods. The analysis of proteins becomes especially challenging when only small sample volumes are available, concomitantly with low concentrations of proteins. Time critical situations pose additional challenges. Due to these challenges, conventional macro-scale separation techniques reach their limitations. While

Rapid and reliable separation and analysis of proteins require powerful analytical methods. The analysis of proteins becomes especially challenging when only small sample volumes are available, concomitantly with low concentrations of proteins. Time critical situations pose additional challenges. Due to these challenges, conventional macro-scale separation techniques reach their limitations. While microfluidic devices require only pL-nL sample volumes, they offer several advantages such as speed, efficiency, and high throughput. This work elucidates the capability to manipulate proteins in a rapid and reliable manner with a novel migration technique, namely dielectrophoresis (DEP). Since protein analysis can often be achieved through a combination of orthogonal techniques, adding DEP as a gradient technique to the portfolio of protein manipulation methods can extend and improve combinatorial approaches. To this aim, microfluidic devices tailored with integrated insulating obstacles were fabricated to create inhomogeneous electric fields evoking insulator-based DEP (iDEP). A main focus of this work was the development of pre-concentration devices where topological micropost arrays are fabricated using standard photo- and soft lithographic techniques. With these devices, positive DEP-driven streaming of proteins was demonstrated for the first time using immunoglobulin G (IgG) and bovine serum albumin. Experimentally observed iDEP concentrations of both proteins were in excellent agreement with positive DEP concentration profiles obtained by numerical simulations. Moreover, the micropost iDEP devices were improved by introducing nano-constrictions with focused ion beam milling with which numerical simulations suggested enhancement of the DEP effect, leading to a 12-fold increase in concentration of IgG. Additionally, concentration of β-galactosidase was observed, which seems to occur due to an interplay of negative DEP, electroosmosis, electrokinesis, diffusion, and ion concentration polarization. A detailed study was performed to investigate factors influencing protein DEP under DC conditions, including electroosmosis, electrophoresis, and Joule heating. Specifically, temperature rise within the iDEP device due to Joule heating was measured experimentally with spatial and temporal resolution by employing the thermosensitive dye Rhodamine B. Unlike DNA and cells, protein DEP behavior is not well understood to date. Therefore, this detailed study of protein DEP provides novel information to eventually optimize this protein migration method for pre-concentration, separation, and fractionation.
ContributorsNakano, Asuka (Author) / Ros, Alexandra (Thesis advisor) / Hayes, Mark (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2014
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Description
Photosynthesis is the primary source of energy for most living organisms. Light harvesting complexes (LHC) play a vital role in harvesting sunlight and passing it on to the protein complexes of the electron transfer chain which create the electrochemical potential across the membrane which drives ATP synthesis. phycobilisomes (PBS) are

Photosynthesis is the primary source of energy for most living organisms. Light harvesting complexes (LHC) play a vital role in harvesting sunlight and passing it on to the protein complexes of the electron transfer chain which create the electrochemical potential across the membrane which drives ATP synthesis. phycobilisomes (PBS) are the most important LHCs in cyanobacteria. PBS is a complex of three light harvesting proteins: phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC). This work has been done on a newly discovered cyanobacterium called Leptolyngbya Heron Island (L.HI). This study has three important goals: 1) Sequencing, assembly and annotation of the L.HI genome - Since this is a newly discovered cyanobacterium, its genome was not previously elucidated. Illumina sequencing, a type of next generation sequencing (NGS) technology was employed to sequence the genome. Unfortunately, the natural isolate contained other contaminating and potentially symbiotic bacterial populations. A novel bioinformatics strategy for separating DNA from contaminating bacterial populations from that of L.HI was devised which involves a combination of tetranucleotide frequency, %(G+C), BLAST analysis and gene annotation. 2) Structural elucidation of phycoerythrin - Phycoerythrin is the most important protein in the PBS assembly because it is one of the few light harvesting proteins which absorbs green light. The protein was crystallized and its structure solved to a resolution of 2Å. This protein contains two chemically distinct types of chromophores: phycourobilin and phycoerythrobilin. Energy transfer calculations indicate that there is unidirectional flow of energy from phycourobilin to phycoerythrobilin. Energy transfer time constants using Forster energy transfer theory have been found to be consistent with experimental data available in literature. 3) Effect of chromatic acclimation on photosystems - Chromatic acclimation is a phenomenon in which an organism modulates the ratio of PE/PC with change in light conditions. Our investigation in case of L.HI has revealed that the PE is expressed more in green light than PC in red light. This leads to unequal harvesting of light in these two states. Therefore, photosystem II expression is increased in red-light acclimatized cells coupled with an increase in number of PBS.
ContributorsPaul, Robin (Author) / Fromme, Petra (Thesis advisor) / Ros, Alexandra (Committee member) / Roberson, Robert (Committee member) / Arizona State University (Publisher)
Created2014
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Description
Membrane proteins are a vital part of cellular structure. They are directly involved in many important cellular functions, such as uptake, signaling, respiration, and photosynthesis, among others. Despite their importance, however, less than 500 unique membrane protein structures have been determined to date. This is due to several difficulties with

Membrane proteins are a vital part of cellular structure. They are directly involved in many important cellular functions, such as uptake, signaling, respiration, and photosynthesis, among others. Despite their importance, however, less than 500 unique membrane protein structures have been determined to date. This is due to several difficulties with macromolecular crystallography, primarily the difficulty of growing large, well-ordered protein crystals. Since the first proof of concept for femtosecond nanocrystallography showing that diffraction patterns can be collected on extremely small crystals, thus negating the need to grow larger crystals, there have been many exciting advancements in the field. The technique has been proven to show high spatial resolution, thus making it a viable method for structural biology. However, due to the ultrafast nature of the technique, which allows for a lack of radiation damage in imaging, even more interesting experiments are possible, and the first temporal and spatial images of an undamaged structure could be acquired. This concept was denoted as time-resolved femtosecond nanocrystallography.

This dissertation presents on the first time-resolved data set of Photosystem II where structural changes can actually be seen without radiation damage. In order to accomplish this, new crystallization techniques had to be developed so that enough crystals could be made for the liquid jet to deliver a fully hydrated stream of crystals to the high-powered X-ray source. These changes are still in the preliminary stages due to the slightly lower resolution data obtained, but they are still a promising show of the power of this new technique. With further optimization of crystal growth methods and quality, injection technique, and continued development of data analysis software, it is only a matter of time before the ability to make movies of molecules in motion from X-ray diffraction snapshots in time exists. The work presented here is the first step in that process.
ContributorsKupitz, Christopher (Author) / Fromme, Petra (Thesis advisor) / Spence, John C. (Thesis advisor) / Redding, Kevin (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2014
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Description
Ribulose-1, 5-bisphosphate carboxylase oxygenase, commonly known as RuBisCO, is an enzyme involved in carbon fixation in photosynthetic organisms. The enzyme is subject to a mechanism-based deactivation during its catalytic cycle. RuBisCO activase (Rca), an ancillary enzyme belonging to the AAA+ family of the ATP-ases, rescues RuBisCO by facilitating the removal

Ribulose-1, 5-bisphosphate carboxylase oxygenase, commonly known as RuBisCO, is an enzyme involved in carbon fixation in photosynthetic organisms. The enzyme is subject to a mechanism-based deactivation during its catalytic cycle. RuBisCO activase (Rca), an ancillary enzyme belonging to the AAA+ family of the ATP-ases, rescues RuBisCO by facilitating the removal of the tightly bound sugar phosphates from the active sites of RuBisCO. In this work, we investigated the ATP/ADP dependent oligomerization equilibrium of fluorescently tagged Rca for a wide range of concentrations using fluorescence correlation spectroscopy. Results show that in the presence of ADP-Mg2+, the oligomerization state of Rca gradually changes in steps of two subunits. The most probable association model supports the dissociation constants (K_d) of ∼4, 1, 1 μM for the monomer-dimer, dimer-tetramer, and tetramer-hexamer equlibria, respectively. Rca continues to assemble at higher concentrations which are indicative of the formation of aggregates. In the presence of ATP-Mg2+, a similar stepwise assembly is observed. However, at higher concentrations (30-75 µM), the average oligomeric size remains relatively unchanged around six subunits per oligomer. This is in sharp contrast with observations in ADP-Mg2+, where a marked decrease in the diffusion coefficient of Rca was observed, consistent with the formation of aggregates. The estimated K_d values obtained from the analysis of the FCS decays were similar for the first steps of the assembly process in both ADP-Mg2+ and ATP-Mg2+. However, the formation of the hexamer from the tetramer is much more favored in ATP-Mg2+, as evidenced from 20 fold lower K_d associated with this assembly step. This suggests that the formation of a hexameric ring in the presence of ATP-Mg2+. In addition to that, Rca aggregation is largely suppressed in the presence of ATP-Mg2+, as evidenced from the 1000 fold larger K_d value for the hexamer-24 mer association step. In essence, a fluorescence-based method was developed to monitor in vitro protein oligomerization and was successfully applied with Rca. The results provide a strong hint at the active oligomeric structure of Rca, and this information will hopefully help the ongoing research on the mechanistic enzymology of Rca.
ContributorsChakraborty, Manas (Author) / Levitus, Marcia (Thesis advisor) / Angell, Charles (Committee member) / Lindsay, Stuart (Committee member) / Arizona State University (Publisher)
Created2014
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Description
Biophysical techniques have been increasingly applied toward answering biological questions with more precision. Here, three different biological systems were studied with the goal of understanding their dynamic differences, either conformational dynamics within the system or oligomerization dynamics between monomers. With Cy3 on the 5' end of DNA, the

Biophysical techniques have been increasingly applied toward answering biological questions with more precision. Here, three different biological systems were studied with the goal of understanding their dynamic differences, either conformational dynamics within the system or oligomerization dynamics between monomers. With Cy3 on the 5' end of DNA, the effects of changing the terminal base pair were explored using temperature-dependent quantum yields. It was discovered, in combination with simulations, that a terminal thymine base has the weakest stacking interactions with the Cy3 dye compared to the other three bases. With ME1 heterodimers, the goal was to see if engineering a salt bridge at the dimerization interface could allow for control over dimerization in a pH-dependent manner. This was performed experimentally by measuring FRET between monomers containing either a Dap or an Asp mutation and comparing FRET efficiency at different pHs. It was demonstrated that the heterodimeric salt bridge would only form in a pH range near neutrality. Finally, with DNA processivity clamps, one aim was to compare the equilibrium dissociation constants, kinetic rate constants, and lifetimes of the closed rings for beta clamp and PCNA. This was done using a variety of biophysical techniques but with three as the main focus: fluorescence correlation spectroscopy, single-molecule experiments, and time-correlated single photon counting measurements. The stability of beta clamp was found to be three orders of magnitude higher when measuring solution stability but only one order of magnitude higher when measuring intrinsic stability, which is a result of salt bridge interactions in the interface of beta clamp. Ongoing work built upon the findings from this project by attempting to disrupt interface stability of different beta clamp mutants by adding salt or changing the pH of the solution. Lingering questions about the dynamics of different areas of the clamps has led to another project for which we have developed a control to demystify some unexpected similarities between beta clamp mutants. With that project, we show that single-labeled and double-labeled samples have similar autocorrelation decays in florescence correlation spectroscopy, allowing us to rule out the dyes themselves as causing fluctuations in the 10-100 microsecond timescale.
ContributorsBinder, Jennifer (Author) / Levitus, Marcia (Thesis advisor) / Wachter, Rebekka (Committee member) / Ros, Robert (Committee member) / Arizona State University (Publisher)
Created2015
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Description
The communication of genetic material with biomolecules has been a major interest in cancer biology research for decades. Among its different levels of involvement, DNA is known to be a target of several antitumor agents. Additionally, tissue specific interaction between macromolecules such as proteins and structurally important regions of DNA

The communication of genetic material with biomolecules has been a major interest in cancer biology research for decades. Among its different levels of involvement, DNA is known to be a target of several antitumor agents. Additionally, tissue specific interaction between macromolecules such as proteins and structurally important regions of DNA has been reported to define the onset of certain types of cancers.

Illustrated in Chapter 1 is the general history of research on the interaction of DNA and anticancer drugs, most importantly different congener of bleomycin (BLM). Additionally, several synthetic analogues of bleomycin, including the structural components and functionalities, are discussed.

Chapter 2 describes a new approach to study the double-strand DNA lesion caused by antitumor drug bleomycin. The hairpin DNA library used in this study displays numerous cleavage sites demonstrating the versatility of bleomycin interaction with DNA. Interestingly, some of those cleavage sites suggest a novel mechanism of bleomycin interaction, which has not been reported before.

Cytidine methylation has generally been found to decrease site-specific cleavage of DNA by BLM, possibly due to structural change and subsequent reduced bleomycin-mediated recognition of DNA. As illustrated in Chapter 3, three hairpin DNAs known to be strongly bound by bleomycin, and their methylated counterparts, were used to study the dynamics of bleomycin-induced degradation of DNAs in cancer cells. Interestingly, cytidine methylation on one of the DNAs has also shown a major shift in the intensity of bleomycin induced double-strand DNA cleavage pattern, which is known to be a more potent form of bleomycin induced cleavages.

DNA secondary structures are known to play important roles in gene regulation. Chapter 4 demonstrates a structural change of the BCL2 promoter element as a result of its dynamic interaction with the individual domains of hnRNP LL, which is essential to facilitate the transcription of BCL2. Furthermore, an in vitro protein synthesis technique has been employed to study the dynamic interaction between protein domains and the i-motif DNA within the promoter element. Several constructs were made involving replacement of a single amino acid with a fluorescent analogue, and these were used to study FRET between domain 1 and the i-motif, the later of which harbored a fluorescent acceptor nucleotide analogue.
ContributorsRoy, Basab (Author) / Hecht, Sidney M. (Thesis advisor) / Jones, Anne (Committee member) / Levitus, Marcia (Committee member) / Chaput, John (Committee member) / Arizona State University (Publisher)
Created2014
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Description
The AAA+ ATPase Rubisco activase (Rca) regulates the activity of Rubisco, the photosynthetic enzyme responsible for catalyzing biological carbon fixation. However, the detailed mechanism by which Rca self-association controls Rubisco reactivation activity remains poorly understood. In this work, we are using fluorescence correlation spectroscopy (FCS) to better characterize the thermodynamics

The AAA+ ATPase Rubisco activase (Rca) regulates the activity of Rubisco, the photosynthetic enzyme responsible for catalyzing biological carbon fixation. However, the detailed mechanism by which Rca self-association controls Rubisco reactivation activity remains poorly understood. In this work, we are using fluorescence correlation spectroscopy (FCS) to better characterize the thermodynamics of the assembly process of cotton Rca. We present FCS data for Rca in the presence of Mg*ATPgS and Mg*ADP and for the D173N Walker B motif mutant in the presence of Mg*ATP. Our data are consistent with promotion and stabilization of hexamers by Mg*ATPgS and Mg*ATP, whereas Mg*ADP facilitates continuous assembly. We find that in the presence of Mg·ADP, Rca self-associates in a step-wise fashion to form oligomeric and higher order forms, with a strong size dependence on subunit concentration. The monomer is the dominant species below 0.5 micromolar, whereas the hexamer appears to be most populated in the 10-30 micromolar range. Large assemblies containing on the order of 24 subunits become dominant above 40 micromolar, with continued assembly at even higher concentrations. Our data are consistent with a highly dynamic exchange of subunits among oligomeric species of diverse sizes. The most likely ADP-mediated assembly mechanism seems to involve the formation of spiral supra-molecular structures that grow along the helical axis by the step-wise addition of dimeric units. To examine the effect of Mg·ATP on oligomerization, we have generated the D173N mutant of Rca, which binds but does not hydrolyze ATP. In range of 8 and 70 micromolar, 60-80% of Rca is predicted to form hexamers in the presence of Mg*ATP compared to just 30-40% with Mg*ADP. We see a clear trend at which hexamerization occurs at high ATP:ADP ratios and in addition, at increasing concentrations of free magnesium ions to 5 milimolar that results in formation of six subunits. We present an assembly model where Mg*ATP promotes and stabilizes hexamerization at low micromolar Rca concentrations relative to Mg*ADP, and suggest that this results from closed ring hexamer formation in Mg*ATP and open hexameric spiral formation in Mg*ADP .
ContributorsKuriata, Agnieszka (Author) / Wachter, Rebekka (Thesis advisor) / Redding, Kevin (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2014
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Description
In an effort to begin validating the large number of discovered candidate biomarkers, proteomics is beginning to shift from shotgun proteomic experiments towards targeted proteomic approaches that provide solutions to automation and economic concerns. Such approaches to validate biomarkers necessitate the mass spectrometric analysis of hundreds to thousands of human

In an effort to begin validating the large number of discovered candidate biomarkers, proteomics is beginning to shift from shotgun proteomic experiments towards targeted proteomic approaches that provide solutions to automation and economic concerns. Such approaches to validate biomarkers necessitate the mass spectrometric analysis of hundreds to thousands of human samples. As this takes place, a serendipitous opportunity has become evident. By the virtue that as one narrows the focus towards "single" protein targets (instead of entire proteomes) using pan-antibody-based enrichment techniques, a discovery science has emerged, so to speak. This is due to the largely unknown context in which "single" proteins exist in blood (i.e. polymorphisms, transcript variants, and posttranslational modifications) and hence, targeted proteomics has applications for established biomarkers. Furthermore, besides protein heterogeneity accounting for interferences with conventional immunometric platforms, it is becoming evident that this formerly hidden dimension of structural information also contains rich-pathobiological information. Consequently, targeted proteomics studies that aim to ascertain a protein's genuine presentation within disease- stratified populations and serve as a stepping-stone within a biomarker translational pipeline are of clinical interest. Roughly 128 million Americans are pre-diabetic, diabetic, and/or have kidney disease and public and private spending for treating these diseases is in the hundreds of billions of dollars. In an effort to create new solutions for the early detection and management of these conditions, described herein is the design, development, and translation of mass spectrometric immunoassays targeted towards diabetes and kidney disease. Population proteomics experiments were performed for the following clinically relevant proteins: insulin, C-peptide, RANTES, and parathyroid hormone. At least thirty-eight protein isoforms were detected. Besides the numerous disease correlations confronted within the disease-stratified cohorts, certain isoforms also appeared to be causally related to the underlying pathophysiology and/or have therapeutic implications. Technical advancements include multiplexed isoform quantification as well a "dual- extraction" methodology for eliminating non-specific proteins while simultaneously validating isoforms. Industrial efforts towards widespread clinical adoption are also described. Consequently, this work lays a foundation for the translation of mass spectrometric immunoassays into the clinical arena and simultaneously presents the most recent advancements concerning the mass spectrometric immunoassay approach.
ContributorsOran, Paul (Author) / Nelson, Randall (Thesis advisor) / Hayes, Mark (Thesis advisor) / Ros, Alexandra (Committee member) / Williams, Peter (Committee member) / Arizona State University (Publisher)
Created2011