Chemistry

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Open Access
A Comparative Study Performed on Two Ambient Ionization Techniques: Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) and Easy Ambient Sonic Spray Ionization Mass Spectrometry (EASI-MS)
(2015-08-28) Hamid, Tanam Sanjana; Ifa, Demian R.
Main project of my master’s thesis involved comparison between two sister methods: desorption electrospray ionization mass spectrometry (DESI−MS) and easy ambient ionization mass spectrometry (EASI−MS). These methods are compared in terms of spatial resolution, limits of detection and imaging capabilities. Their applicability in forensic science was further explored by comparing the MS/MS imaging capabilities. Under the same experimental conditions, we found that both DESI and EASI−MS have similar spray spot size resulting in similar spatial resolution. For the limits of detection (LOD) experiment, the LOD of DESI−MS was found to be lower than EASI−MS by an order of magnitude. When we compared the mass spectra and the chemical images of DESI and EASI, we found that both methods produce similar chemical specificity. When the spectra were carefully inspected, a reduction in signal intensity was observed for EASI−MS compared to DESI−MS demonstrating that the sensitivity of DESI−MS is higher than EASI−MS. Other projects involved characterization of zebra fish tissue system made up of bile salts, phospholipids and fatty acids. By mapping the spatial distribution of these ions, 2D chemical images of organs such as the stomach, the nervous system and the whole body zebra fish were created. I also studied if matrix can impact ionization of proteins by DESI−MS. The aim was to improve the poor desorption of proteins from the surface of DESI−MS. We concluded that the matrix neither increase nor decreases the ionization efficiency of proteins by DESI−MS.
Open Access
A Desorption Electrospray Ionization Mass Spectrometry Imaging Approach to Monitor Toxic Ionic Liquids in Zebrafish (Danio Rerio)
(2016-11-25) Perez, Consuelo Javiera; Ifa, Demian R.
Ambient mass spectrometry imaging has become an increasingly powerful technique for the direct analysis of biological tissues in the open environment with minimal sample preparation and fast analyses times. In this study, we introduce desorption electrospray ionization mass spectrometry imaging (DESI-MSI) as a novel, rapid and sensitive approach to monitor toxic ionic liquids in zebrafish (Danio rerio). AMMOENG 130, distearyldimethylammonium chloride, is used as a surfactant in commercialized personal care products. AMMOENG 130 is toxic to zebrafish exhibiting an LC50 of 5.2 mg/L, causing extensive damage to gill secondary lamellae and increasing membrane permeability. Zebrafish were exposed to AMMOENG 130 in a static 96 hour acute toxicity study in concentrations below and near the LC50 ranging from 1.25-5.0 mg/L. DESI-MS analysis of zebrafish gills revealed the appearance of a metabolite in all concentrations of exposure. The metabolite was further characterized with a high resolution hybrid LTQ-Oritrap mass spectrometer as the stearyltrimethylammonium ion of m/z 312. Whole body zebrafish tissue from exposure concentrations were mapped for AMMOENG 130 and its dealkylated metabolite, accumulation was mainly found in the nervous and respiratory systems suggesting that AMMOENG 130 and its metabolite were capable of penetrating the blood brain barrier of zebrafish brain uncovering potential neurotoxic effects. To evaluate DESI-MS as a potential environmental IL monitoring tool, AMMOENG 130 was spotted in varying concentrations onto PTFE surfaces and the limit of detection found was 10 g/L. Hence, we demonstrate here the simultaneous characterization, accumulation and metabolism of a toxic IL in whole body zebrafish analyzed by DESI-MSI. This ambient ionization mass spectrometry technique shows great promise for the direct analysis of biological tissues to monitor toxic and persistent environmental pollutants in aquatic organisms for qualitative analysis with future potential for quantitative analysis.
Open Access
A Novel Ground Based Mass-Balance Method for Methane Emission Quantification
(2018-08-27) Fujs, William Ivan; McLaren, Robert
Emission of CH4 from landfills in Canada are not well constrained and in Ontario constitute the largest point source emitters. The purpose of this study was to develop and test a ground based method for quantifying CH4 emissions from large sources. Emissions of CH4 were quantified from the Keele Valley Landfill (KVL) using ground based mobile mass balance approach where a mobile cavity ring down spectroscopy (CRDS) instrument captured the downwind field of CH4 mixing ratio enhancements relative to the background. The approach involves measuring the downwind field of enhancements at successively further distances from the source until the integrated CH4 enhancements converge. On multiple days in April and May 2016 multiple transects were driven upwind and at increasing distances downwind from the KVL with the CRDS in a vehicle in order to determine integral flux emission estimates. The KVL was found to be a major local source of CH4 even though CH4 collection used for electricity generation is now terminated. An average emission rate of 429 199 kg/hr of CH4 was measured in 2017 on several days, which is less than the ECCC emission inventory value of 2149 kg/hr [2015]. The source of the discrepancy is not fully understood, but may be related to the shutdown of the KVL facility. The largest source of uncertainty in our emission estimate calculation was the height of the PBL, which
Open Access
A Stable Dicationic Salt in Reactive DESI-MS Imaging in Positive Ion Mode to Analyze Biological Samples
(2015-12-16) Lostun, Dragos; Ifa, Demian R.
A dicationic salt was applied for imaging in reactive desorption electrospray ionization mass spectrometry (DESI-MS) on biological samples. The samples interrogated in this experiment were whole body zebrafish tissues and rat brain tissue. The stable dictation salt forms a stable bond with biological tissue fatty acids and lipids and allows detection in positive ion mode. Reactive DESI-MS imaging in positive ion mode of rat brain and zebrafish tissues allowed enhanced detection of certain compounds commonly observed in negative ion mode. Variance in intensity between negative and positive mode indicate that the ionization process could be affected by matrix effects of various tissues, leading to different overall intensity which could provide new information on the same compounds when compared between negative ion mode images and reactive DESI positive ion mode images.
Open Access
A Study of Bis (Amido) Pyridine Coordination Chemistry to Ruthenium and Cobalt Metals
(2018-08-27) Hana, Faidh; Lavoie, Gino
This thesis presents the work to study and expand the coordination chemistry of bis(amido)pyridine to ruthenium and cobalt. Chapter 1 presents the properties, the coordination chemistry and the synthesis of the bis(amido)pyridine ligand. Chapter 2 focuses on experimental procedures to synthesize bis(amido)pyridine-supported ruthenium complexes. Chapter 3 focuses on the coordination of the ligand to cobalt(III) where a series of low-valent Co(III) square planar complexes supported by the dianionic ligand is reported.
Open Access
Adsorption and Self-assembly of Proteins at Chemically Modified Surfaces
(2015-08-28) Zahedijasbi, Shohreh; Morin, Sylvie
Our research is mostly focused on the subject of adsorption and self-assembly of proteins at modified surfaces. We employed atomic force microscopy (AFM) to study bovine serum albumin (BSA) and insulin adsorption at 1-decene- and methyl 10-undecenoate-modified Si(111) substrates. The results were compared to matrix-assisted laser desorption/ionization-mass spectroscopy (MALDI-MS) data previously published in the Morin group. The MALDI-MS spectra showed a big insulin signal, while BSA was not detected for 1 mg/mL BSA and 10% saturated insulin solutions. Our AFM images revealed considerable BSA adsorption, whereas it was not possible to observe insulin molecules. This clearly shows that in addition to the low quantity of the protein, a stronger interaction between the surface and the protein may result in a weak MALDI-MS signal and prevent quantitative analysis using this technique. Self-assembled monolayers on Au surfaces were used to investigate the oligomerization of truncated K122-4 (ΔK122-4) pilins into a new protein nanostructure. Employing AFM, we observed that at hydrophobic layers, with more than 10-30% accessible hydrophobic component, ΔK122-4 pilins oligomerize to a nanoweb. To differentiate the structure of ΔK122-4 nanoweb from the aggregation of the pilins at hydrophilic surfaces, electrochemical impedance spectroscopy was employed. Through equivalent circuit fitting, capacitance values of 3.7 ± 0.4 μF/cm2 and 2.5 ± 0.2 μF/cm2 were obtained for the aggregated pilins and the web pattern, respectively. Because of the higher average thickness of the former layer, this could support the presence of water in this layer. A dielectric constant (ε) of 5.1 ± 0.7 was obtained for the nanoweb. This low value could indicate a more compact and ordered assembly. Finally, conductive protein nanotube (PNTs) fabrication was performed by electroless copper deposition at ΔK122-4 PNTs in aqueous solution. The nanostructures were catalyzed by PdCl42-. At least 20% of the accessible amino acids of the PNTs have S-, N- and O-donor side chains which are able to form a complex with Pd(II). Metallization was verified using scanning electron microscopy (SEM).
Open Access
Advancing Kinetic Capillary Electrophoresis for High-Efficiency Screening of Oligonucleotide Libraries in Drug Discovery
(2024-03-16) Le, An Thi Hoai; Krylov, Sergey N.
Identifying protein binders is the first step in drug discovery. The combinatorial approach, in which a library of compounds is subjected to affinity screening against a target protein, is a major way for identifying protein binders. Oligonucleotide libraries constitute the largest source of material for such affinity screening. Selecting protein binders from such libraries requires a highly efficient method for separation of protein−oligonucleotide complexes from the excess of unbound oligonucleotides. Kinetic Capillary Electrophoresis (KCE) is a rapidly advancing technique in affinity applications. It reportedly has superior partitioning efficiency, but screening oligonucleotide libraries by KCE has many challenges which must be addressed before KCE can compete with conventional surface-based screening. The goal of my research is to transform KCE into a versatile technology for screening protein binders from oligonucleotide libraries. To overcome the nonbinder background issue in KCE-based partitioning, I introduce Ideal-Filter Capillary Electrophoresis (IFCE), where binders and nonbinders travel in opposite directions. While successfully implementing IFCE conditions to be compatible with physiological buffers, a remarkable partitioning efficiency of 109 is achieved, the highest recorded so far. Further, I develop the first quantitative characterization of all KCE-based partitioning modes for a diverse range of protein target sizes. This systematic analysis provides guidance for CE users on selecting appropriate KCE-based partitioning conditions for a given protein target. Next, I conduct the first experimental investigation into the influence of target concentration on binder selection, aiding researchers in identifying an appropriate range of target concentrations to prevent selection failures. Finally, I gather insights from all these works to demonstrate the first highly efficient KCE-based selection of protein binders from DNA-encoded library of small molecules (DEL). This pioneering achievement showcases the capabilities of KCE-based partitioning within the context of DEL-based drug discovery, marking a significant advancement in the field.
Open Access
Alkylidene Dihydropyridines as Useful Intermediates for Functionalization of 4-Alkylpyridines
(2024-03-16) Shi, Jiaqi; Orellana, Arturo
Pyridines are widely used and extensively studied heterocyclic compounds in industry and academia. Developing mild and selective methods for functionalizing pyridines would facilitate the synthesis of more structurally diverse pyridine derivatives, which could aid the development of potential drug candidates and streamline the drug discovery process. Through a “soft-enolization” logic, 4-alkylpyridines can be readily converted to corresponding alkylidene dihydropyridines (ADHPs), enabling mild and selective palladium-catalyzed allylation and dehydrogenation, as previously shown by our group. During the mechanistic study of the allylation reaction, prompted by the low enantioselectivity of pyridine allylation using optically active ligands, our group demonstrated that ADHPs are “soft” nucleophiles towards the allylpalladium(II) complex. We thus began employing this class of intermediates as soft nucleophiles in other reactions. Here I will first discuss the work towards enantioselective allylation of 4-alkylpyridines. Then I will describe the conjugate addition of ADHPs to a,b-unsaturated ketones that are activated by triethylsilyl triflate. Finally, I will introduce the approach to unite piperidine and pyridine with a carbon atom through addition of ADHPs to protonated pyridines with subsequent transfer hydrogenation reaction.
Open Access
Ambient Ionization: Surface Analysis of Sexual Assault Evidence and 2-Dimensional Imaging of Whole-Body Zebra Fish (Danio Rerio) Using Desorption Electrospray Ionization
(2015-08-28) Chramow, Alexander; Ifa, Demian R.
Desorption electrospray ionization (DESI) is an ambient surface analysis technique capable of producing 2D and 3D images. The ionization mechanism utilizes a pneumatically assisted sprayer to deposit a charged solvent onto a sample surface. Subsequent impacting primary droplets produce ejecting surface secondary droplets containing desolved analytes, which are then detected by a mass analyzer. This thesis explores two fields of application of DESI, forensics and biological tissue analysis. The former involves the analysis of sexual assault evidence, in the form of condoms, lubricants, and their residues as a potential aid in convicting perpetrators. The latter focuses on investigating the potential use of the zebra fish (Danio rerio) as a model vertebrate organism for future toxicological and biological research. Whole-body 2D images were created, highlighting areas of interest such as, the brain, spinal cord, liver, and body fat.
Open Access
An Evaluation of the Contributions of Short and Long Range Pollutant Transport on Southern Ontario's Atmospheric Sulfur Content
(2023-08-04) Pritchard-Oh, Austin Michael; McLaren, Robert
Sulfur dioxide (SO2), particle sulfate (p-SO4-2), and total sulfur (SOx) were evaluated at York University, in Toronto, Canada, using a ThermoFisher SO2 analyzer and an Aerodyne Aerosol Mass Spectrometer. These measurements were compared with a dataset from 2002-2020, which demonstrated a downward trend in atmospheric sulfur. Toronto’s atmospheric sulfur decline has plateaued in recent years. Current mean SO2 was 0.37 ppb, p-SO4-2 was 0.60 μg/m3, and SOx was 1.6 μg/m3. There were no significant seasonal or daytime/nighttime differences. The effects of pollutant transport and major contributors to Toronto’s sulfur were determined. Major contributors to SO2 included Hamilton metal refineries, Greater Toronto Area chemical production facilities, the Nanticoke petroleum refinery, and vehicle emissions. Major contributors to p-SO4-2 are Sarnia petroleum and chemical production, Sudbury metal refineries, and long-range transport from the Ohio River Valley. A field study mass-balance determination of Sarnia’s SO2 emissions was estimated at 0.18 tonnes/hr (4,300 kg/day).
Open Access
Application of Pd-NHC Complexes in Challenging Amination Reactions
(2019-07-02) Sharif, Sepideh; Organ, Michael George
Among Pd-catalyzed carbon-heteroatom cross-coupling reactions, C-N bond formation, known as Buchwald-Hartwig amination (BHA), is by far the most studied reaction owing to the prevalence of arylated amines in pharmaceuticals, natural products, organic materials, and catalysts. Despite the tremendous progress that has been achieved with respect to improving the efficiency and expanding the scope of BHA of (hetero)aryl halides and amine nucleophilic partners, some challenges remain to be addressed. These challenges include selective monoarylation of primary alkyl amines, enantioretentive N-arylation of -amino esters and arylation of amide nucleophiles. This research is mainly focused on evaluating the reactivity of Pd-NHC pre-catalysts, in particular Pd-PEPPSI (Pyridine Enhanced Pre-catalyst Preparation Stabilization and Initiation) complexes in such challenging BHA reactions. Pd-PEPPSI-IPentCI, was identified as one of the most reactive and selective pre-catalysts yet reported in the literature in the arylation of primary alkyl amines. The high level of selectivity that was exhibited by Pd-PEPPSI-IPentCI is assured by the use of the mild, soluble and sterically demanding sodium salt of butylated hydroxytoluene (NaBHT) as the base in this transformation. PdPEPPSIIPentClopicoline was shown to effectively couple a variety of amino acids as the tertbutyl ester with heteroaryl chlorides in high yields and with excellent stereoretention of the acidic proton adjacent to the ester. Control experiments revealed that racemization is basemediated, with no evidence of Pdmediated hydride elimination, and that racemization occurs only after the product is formed. Studies also revealed that increasing the steric bulk of the ester moiety on the amino acid (e.g., ethyl to tertbutyl) drastically slows racemization of the product. Boron-derived Lewis acids such as (secBu)3B, Et3B, and BCF have been shown to effectively promote the coupling of amide nucleophiles to a wide variety of oxidative addition partners using (DiMeIHeptCl)Pd(cinammyl)Cl. Through a combination of NMR spectroscopy and control studies with and without oxygen and radical scavengers, we propose that boron-imidates form under the basic reaction conditions that aid coordination of nitrogen to PdII, which is rate limiting, and directly delivers the intermediate for reductive elimination. During the course of optimization of aryl amidation, we found the hydrodehalogenated arene as the side product when NaBHT was employed as base. Extensive control experiments revealed that NaBHT can serve as a hydride delivering agent in Pd-catalyzed hydrodehalogenation of (hetero)aryl halides. During the course this study, the structure of the phenolate was found to be critical to the success of this protocol, that is, bulky di-ortho- substituents on phenolate are required to achieve optimal results.
Open Access
Aryl-Substituted Imino-N-Heterocyclic Carbene Complexes Of Late Transition Metals: Synthesis and Reactivity Studies
(2015-01-26) Badaj, Anna Candace; Lavoie, Gino G
N-heterocyclic carbenes (NHCs) have found enormous success as ancillary ligands in catalysts in many areas of organometallic chemistry. Surprisingly, their use in olefin polymerization, until recently, was not widely explored. The focus of this thesis is to investigate the synthesis of a bidentate ligand scaffold that incorporates an NHC moiety and to study the ability of the resulting complexes to catalyze chemical transformations such as olefin polymerization. The synthesis and characterization of several N-aryl substituted imino-imidazolin-2-ylidene (C^ImineR) ligand precursors was achieved following one of two synthetic protocols. Coordination of these ligands was explored with Group 11 metals in order to develop synthetic protocols which were later extended to prepare complexes of ruthenium, cobalt, iron, nickel, palladium and zinc. The coordination of C^ImineR ligands to nickel using new copper carbene dimers as the transmetalating agent was established. All the nickel complexes were structurally characterized and the size of the iminic carbon substituent was found to have a profound impact on the bond angles and bond lengths around the metal center. However, when tested for ethylene polymerization activity at standard temperature and pressure, the nickel complexes were found to be inactive. With the discovery the nickel complexes of these C^ImineR ligands were inactive for ethylene polymerization, the research focus was extended to the diamagnetic palladium-methyl complexes in order to gain insight into their thermal stability and insertion chemistry. The nature of the iminic substituent profoundly affects the thermal stability of the neutral palladium complexes. While inactive for ethylene polymerization, these palladium methyl complexes react with CO and isocyanides to form various coordination and insertion products.
Open Access
Bio-Orthogonal Manipulation of Cellular Membranes for Biological Applications
(2018-05-28) O'Brien, Paul J.; Yousaf, Muhammad
Recent breakthroughs using novel chemistry and nanotechnology methodologies for cellular biology applications have spurred tremendous interest in the development of new technologies to manipulate cell-lines or cellular products for biotechnology applications, without complicated or permanent techniques used in academia and industry. Small molecule drugs, proteins, deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) have been employed to manipulate and probe cellular behaviour with the intention of elucidating healthy and diseased molecular pathways for understanding human health. This research focused on the application of liposomal delivery of bioorthogonal chemoselective chemistry to cellular membranes to control biological functions. These smart membranes were used to investigate cellular adhesion, cell spheroid aggregation kinetics and incorporation of ligands. In Chapters 2 and 3, NMR spectroscopy, oxime reaction kinetics and oxime cell surface engineered cell adhesion was correlated and probed under microfluidic conditions, while the robustness of adhesion and flexibility of cluster and tissue formation was established using live cell technique microfluidics. Using this as a base strategy, we developed a general method for the microfluidic manipulation and CSE of cells for dual labelling and flexible delivery of nucleic acids and small molecule ligands (Paul OBrien et al. 2017, in preparation). In Chapter 4, the bioorthogonal liposomal strategy originally utilized for cell adhesion and cell membrane ligand integration was modified and extended to transfect mammalian cell lines with nucleic acids, limiting the use of cationic charge as a mild and general method to tag and target cells in monocultures and co-cultures. The method was characterized using microscopy, protein production quantification and targeting in complex co-cultures for selective internalization. Finally, in Chapter 5 a new chemical moiety termed dialdehyde was designed and synthesized for the easy and mild conjugation of primary amine containing molecules. This conjugation strategy was used to deliver small molecule ligands and macromolecules to bacterial and mammalian cell membranes using a generalized liposomal formation method. The dialdehyde was characterized and modified to contain two dialdehyde moieties with a Polyethylene Glycol (PEG) linker for crosslinking proteins, inorganic surface functionalization, organic bead assembly and cell aggregation for tissue formation.
Open Access
Biomedical Characterization of the WalKR and the LytSR Two Component System From Staphylococcus Aureus
(2015-08-28) Patel, Kevin Hasmukhlal; Golemi-Kotra, Dasantila
Staphylococcus aureus is a major gram-positive pathogen, because of its remarkable capacity to develop resistance against different antimicrobial agents. It has been shown that in S. aureus two component systems (TCS) play an important role in responding to stress induced by antibiotics. Given the regulatory importance of TCS, herein we investigate in vitro the signal transduction mechanism of the WalKR, and the LytSR TCSs. These TCSs play a role in susceptibility towards cell wall active antibiotics and cationic antimicrobial agents, respectively. We found that both kinases, WalK and LytS, are active in vitro and capable of catalyzing ATP-dependent autophosphorylation and subsequent phosphoryl transfer reactions to their cognate response regulators WalR and LytR. In contrast to LytS, the autokinase activity of WalK is highly dependent on K+ and Ca2+ ions. We found that LytR undergoes phosphorylation by small molecule phosphate donors such as acetyl phosphate. Phosphorylation of LytR leads to dimerization of the N-terminal domain. Furthermore, we show that LytR has the ability to bind to the promoter region of lrgAB in its unphosphorylated state. Taken together, these data provide in vitro proof of phosphorylation mediated signal flow in the WalKR and LytSR TCSs.
Open Access
Characterization of Function and Role of FMTA in Staphylococcus Aureus
(2015-08-28) Rahman, Muhammad Masfiqur; Golemi-Kotra, Dasantila
Staphylococcus aureus is one of the major reasons for infectious mortality and morbidity. Due to extensive use of antibiotics, S. aureus has acquired resistance to almost all antibiotics. FmtA is one of the additional factors for methicillin resistance in S. aureus which interacts with wall teichoic acid (WTA) and localizes in the cell division septum. Previous studies showed that FmtA has very weak D-Ala-D-Ala carboxypeptidase (Cpase) activity. In lieu of these findings, we hypothesized that FmtA may require WTA as an activator ligand or protein-protein interactions to become fully functional as Cpase in the division septum. Here we show that WTA is not an activator ligand for Cpase activity of FmtA, but FmtA has an esterase activity on WTA. FmtA can remove D-Ala from D-alanyl ester in WTA. Additionally, we show serine and lysine from sequence motif Ser127-X-X-Lys130 (conserved among penicillin binding proteins (PBPs), β-lactamase and family VIII esterase) of FmtA are involved in this catalytic activity. Mutation studies suggest that both Ser-X-X-Lys motif in FmtA, located at the position S63 and S127 are involved in catalysis. Our results suggest that FmtA’s esterase enzymatic activity depends on both enzyme and substrate concentrations and high concentration of substrate (10 mg/mL WTA) can inhibit the reaction. Beside WTA, FmtA can also remove D-Ala from lipoteichoic acid (LTA). Results from in-vivo studies of WTA from fmtA deletion and fmtA-CM (conditionally over expressed) in S. aureus were in agreement with in-vitro esterase activity. In conclusion, WTA is not an activator ligand for FmtA and that FmtA shows esterase activity towards D-alanyl ester of WTA and LTA. Both SXXK motifs in FmtA are involved in catalysis and Serine and lysine from SXXK motif are important for esterase catalysis. To date, biological function of most esterase is unknown. To our knowledge, this is the first study to report esterase from S. aureus that has sequence similarity to PBPs and β-lactamase and very selective to its biological substrate WTA and LTA compare to synthetic substrate.
Open Access
Characterization of Structures and Dynamics of Intrinsically Disordered Domain and Proteins Using Time Resolved-Hydrogen Deuterium Exchange Mass Spectrometry
(2017-07-27) Zhu, Shaolong; Wilson, Derek J.
Proteins are inherently dynamic. Virtually all of the processes that underlie biological activity, including binding partner recognition, catalysis and allostery among many others, require transient adoption of specific high energy conformations. While many proteins contain highly dynamic regions or Intrinsically Disordered Domains (IDDs), Intrinsically Disordered Proteins (IDPs) are free of well-defined structural features altogether. These domains/proteins play integral roles in a wide variety of biological processes, but are often associated with neurodegenerative disease and cancer. Hence it is crucial to study these biomacromolecules to understand the structural underpinnings of their biological functions as well as the factors that drive disease pathology. However, many of the current high resolution structural techniques (like X-ray crystallography and NMR) are not feasible for such studies because of their reliance on the presence of a well-defined native conformation. An emerging structure labeling technique called Time Resolved-Hydrogen Deuterium Exchange (TR-HDX) can offer an alternative strategy. In this approach, structure-dependent hydrogen/deuterium labelling is measured on the millisecond timescale, providing an exquisitely sensitive picture of the weak hydrogen bonding networks that impart residual structure in IDDs and IDPs. In this work, TR-HDX was implemented onto microfluidic chip to achieve solution phase site-specific analysis of the structures and dynamics of p53 N-terminal transactivation domain and tau protein. Gas-phase TR-HDX was also implemented to study gas-phase protein structures that become populated during Differential Mobility Spectrometry (DMS) ion mobility analyses. Critical advances include the discovery and characterization of an amyloidogenic tau intermediate that may underlie Alzheimers disease pathology, a dynamics-based model for recruitment of co-factors to cancer protein p53 upon phosphorylation, a detailed account of how a set of anti-amyloid drug candidates affect tau structure and dynamics, and development of a method to predict solution-phase protein stability from DMS-HDX measurements.
Open Access
Characterization, Imaging and Quantitation of Small Molecules by Ambient Ionization Mass Spectrometry
(2019-11-22) Prova, Shamina Saiyara; Ifa, Demian R
For more than a century mass spectrometry has been a well-known technique in the field of chemical analytics. Its selectivity and sensitivity has made it popular in various fields. From analysis of pure organics, its use is still being explored in the analysis of biomolecules, either purified or direct from tissue sections. For analyzing these vast arrays of molecules, typically the front end is modulated depending upon the need of the user. For direct analysis of a sample of interest, ionization techniques such as DESI, PS, MALDI, etc are incorporated into the front of the mass spectrometer. In this work, the ambient ionization techniques, DESI-MS and PS-MS was modulated for characterization, imaging and quantification of small molecules. The use of DESI-MS and PS-MS in the field of forensics, microbiology and pharmaceutics is described. With the optimization of the front end modes, better sensitivity, selectively and robustness was ensured.
Open Access
Characterizing Conformational Dynamics and Catalytic Activity of Enzymes by Hydrogen-Deuterium Exchange Mass Spectrometry
(2019-11-22) Knox, Ruth Aurora; Wilson, Derek J
Determining the conformational dynamics of enzymes as they undergo catalysis has been challenging for decades due to the short timescale and limitations of traditional analysis, including techniques such as CPMG 2D NMR and X-ray crystallography. Herein is the study of two proteins: TEM-1 -lactamase, the most common enzyme responsible for the hydrolysis of antibiotics in gram-negative bacteria, and yeast alcohol dehydrogenase (YADH), responsible for facilitating the hydride transfer to NAD+ for energy production in prokaryotes. In the catalysis of both antibiotics and NAD+ reduction the specific residues involved in each binding mode remain under debate. By using a microfluidics workflow and an adjustable reaction volume, time resolved HDX experimentation was used to monitor deuteration events concurrently with catalytic activity on the millisecond to second timescale. Native mass spectrometry enabled studies into binding affinity and monitoring of substrate inactivation on a measured time course. Ion mobility mass spectrometry (IM-MS) was used to provide definitive MS/MS results for protein coverage and provide spatial resolution for all protein/substrate complexes. Additionally, collision induced unfolding (CIU) within the ion mobility cell provides a comparative binding affinity scale for the inhibitory drugs used in the study of TEM-1. Using this wide range of analytical techniques facilitated important discoveries including the isolation of specific residues of TEM-1 mapped to their involvement in different binding modes during catalysis, and subsequently the differentiation in inactivation pathways depending on substrate concentration and type. Further work has isolated residues involved in the turnover event of NAD+ along with residues that display a significant decrease in dynamics with the catalysis of deuterated ethanol compared to the non-deuterated ethanol.
Open Access
Characterizing Protein Dynamics of Protein-Ligand Interactions by Hydrogen-Deuterium Exchange Mass Spectrometry
(2015-01-26) Resetca, Diana; Wilson, Derek J.
The study of protein-ligand and protein-protein interactions is of paramount importance to the understanding of their biological function. Whereas this area of research has been largely dominated by conventional structural biology techniques, such as NMR and X-ray crystallography, an emerging methodology that relies on the implementation of hydrogen deuterium exchange (HDX) powered by MS-based analysis holds the potential to greatly expand on our ability to probe the protein dynamics of fundamental biological processes. In this work, the entire HDX workflow for site-specific analysis of protein dynamics was integrated onto a concerted microfluidic device and applied to the interrogation of the dynamic changes that accompany protein-ligand interactions. This application is described for two model systems: the binding of glutathione (GSH) by Glutathione-S-Transferase (GST), and the binding of three novel salicylic acid-based inhibitors of Signal Transducer and Activator of Transcription 3 (STAT3) to its SH2 domain. This work extends the application of time-resolved electrospray ionization mass spectrometry (TRESI-MS) HDX to the study of protein ligand interaction dynamics and ligand-binding site mapping.
Open Access
Characterizing the Structure-Function Relationship of Pathological Proteins
(2019-07-02) Huang, Xiaojing; Wilson, Derek J.
Protein structure and dynamics dictate the fundamentals of protein function. For example, a structured protein must change its conformation when interacting with various ligands. In addition, misfolded or intrinsically disordered proteins (IDPs) oligomerize into more structured aggregates or amyloid fibrils. Therefore, the study of protein conformations becomes necessary to further understand their cellular functions. By monitoring the exchange of backbone amide hydrogens as a measurement of protein dynamics, time-resolved hydrogen-deuterium exchange (TR HDX) provides advantages in capturing the subtle and transient structural differences over other equilibrium state techniques, such as X-ray crystallography and NMR. Using TR HDX together with other biophysical techniques, we demonstrate the unfolding of lipocalin 2 (Lcn2) in accommodating siderophores. In the second part, we attempt to understand the change in aggregation rate corresponding to conformational changes of polyglutamine expansion protein, huntingtin exon1 (Httex1).