PRISM :: Browsing by Author "Schriemer, David C."

Browsing by Author "Schriemer, David C."

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Open Access
A compositional analysis of membrane microdomains using methods in proteomics
(2011) Khan, Morgan F. S.; Schriemer, David C.; Deans, Julie
Open Access
A novel protein footprinting platform: mass spectrometry of laser-initiated carbene reactions
(2011) Jumper, Chanelle C.; Schriemer, David C.
This work reports a protein labeling method using non-selective carbene reactions of sufficiently high efficiency to permit detection by mass spectrometric methods at the protein, peptide and residue level. The approach uses a diazirine-modified amino acid (L-2-amino-4,4' -azipentanoic acid, "photoleucine") as a label source, which is converted to a highly reactive carbene by pulsed laser photolysis at 355 nm. Labeling of model proteins and peptides was achieved with sensitivity to changes in protein topography brought about by conformational change and ligand binding. Labeling yield is independent of protein concentration over approximately two orders of magnitude, but is weakly dependent on the presence of other chromophores in a photon-limited apparatus. The current configuration required 2 minutes of irradiation for full reagent conversion, however it is shown that comparable yields can be achieved with a single high-energy laser pulse (>100 mJ/pulse, <10 nsec), offering a labeling method with high temporal resolution.
Open Access
Addressing proteolytic efficiency in enzymatic degradation therapy for celiac disease.
(Scientific Reports, 2016-8-2) Rey, M.; Yang, M.L.; Lee, L.; Zhang, Y.; Sheff, J.G.; Sensen, C.W.; Mrazek, H.; Halada, P.; Man, P.; McCarville, J.L.; Verdu, E.F.; Schriemer, David C.
Celiac disease is triggered by partially digested gluten proteins. Enzyme therapies that complete protein digestion in vivo could support a gluten-free diet, but the barrier to completeness is high. Current options require enzyme amounts on the same order as the protein meal itself. In this study, we evaluated proteolytic components of the carnivorous pitcher plant (Nepenthes spp.) for use in this context. Remarkably low doses enhance gliadin solubilization rates, and degrade gliadin slurries within the pH and temporal constraints of human gastric digestion. Potencies in excess of 1200:1 (substrate-to-enzyme) are achieved. Digestion generates small peptides through nepenthesin and neprosin, the latter a novel enzyme defining a previously-unknown class of prolyl endoprotease. The digests also exhibit reduced TG2 conversion rates in the immunogenic regions of gliadin, providing a twin mechanism for evading T-cell recognition. When sensitized and dosed with enzyme-treated gliadin, NOD/DQ8 mice did not show intestinal inflammation, when compared to mice challenged with only pepsin-treated gliadin. The low enzyme load needed for effective digestion suggests that gluten detoxification can be achieved in a meal setting, using metered dosing based on meal size. We demonstrate this by showing efficient antigen processing at total substrate-to-enzyme ratios exceeding 12,000:1.
Open Access
Advancing Techniques of Structural Mass Spectrometry for Integrative Structural Modelling
(2020-04-20) Ziemianowicz, Daniel S.; Schriemer, David C.; Goodarzi, Aaron A.; MacCallum, Justin L.; Fraser, Marie Elizabeth
Proteins are the fundamental functional units underlying all cellular activities. Protein function emerges from structure. To understand cellular activity and the diseases that arise from protein dysfunction we require knowledge of protein structure and structural dynamics. The toolbox offered by mass spectrometry (MS) allows a wide range of perspectives on protein structure, enabled by the application of chemical reagents that can encode structural properties. Improvements in the performance of labelling chemistries in turn can enhance the data returned and the structural models that are ultimately produced. Photogenerated carbenes are one such high-performance chemistry that offer unbiased sampling of protein structure at timescales relevant to protein dynamics. On the other hand, no single method can offer the breadth of data necessary to produce a comprehensive model of protein structure and dynamics—protein systems span broad spatial and temporal scales that exceed the scope of any single technique. To resolve large and complex protein systems, the integration of multiple data sets from orthogonal techniques is necessary. Here, I evaluate and advance structural MS methods with the goal of improving the accuracy and precision of structural models produced by MS-driven integrative structural modelling. Of particular interest is the application of carbene-based crosslinking and covalent labelling reagents which are shown to produce data with greater sequence coverage and improved accuracy in representing the equilibrated conformational state. Novel analytical software routines are developed to overcome the complications that arise from the labelling of proteins with a non-specific chemistry such as ambiguity in localizing modifications. Structural models are produced with integrative modelling workflows, including the development of a novel modelling restraint based on crosslinking and hydrogen/deuterium exchange data. MS-driven integrative modelling is demonstrated on multiple systems, including large complexes and systems with substantial disorder.
Open Access
Assembly of Ebola Virus Matrix Protein VP40 Is Regulated by Latch-Like Properties of N and C Terminal Tails
(PLOS One, 2012-7-5) Silva, Leslie P.; Vanzile, Michael; Bavari, Sina; Aman, J. M. Javad; Schriemer, David C.
The matrix protein VP40 coordinates numerous functions in the viral life cycle of the Ebola virus. These range from the regulation of viral transcription to morphogenesis, packaging and budding of mature virions. Similar to the matrix proteins of other nonsegmented, negative-strand RNA viruses, VP40 proceeds through intermediate states of assembly (e.g. octamers) but it remains unclear how these intermediates are coordinated with the various stages of the life cycle. In this study, we investigate the molecular basis of synchronization as governed by VP40. Hydrogen/deuterium exchange mass spectrometry was used to follow induced structural and conformational changes in VP40. Together with computational modeling, we demonstrate that both extreme N and C terminal tail regions stabilize the monomeric state through a direct association. The tails appear to function as a latch, released upon a specific molecular trigger such as RNA ligation. We propose that triggered release of the tails permits the coordination of late-stage events in the viral life cycle, at the inner membrane of the host cell. Specifically, N-tail release exposes the L-domain motifs PTAP/PPEY to the transport and budding complexes, whereas triggered C-tail release could improve association with the site of budding.
Open Access
Characterizing the Role of the Type VI Secretion System in Interbacterial Species Interactions and Pathogenesis
(2018-12-14) Wong, Megan Jee Quin; Dong, Tao G.; Devinney, Rebekah; Schriemer, David C.
Bacteria require molecular mechanisms to properly sense and respond to their environment. This allows them to compete for specific niches, persist in polymicrobial communities, and contribute to virulence and pathogenesis. One mechanism Gram-negative bacteria have adapted is the use of type VI secretion systems (T6SS). The T6SS is a large needle complex that spans across the entire bacterial cell wall and functions through a contraction mechanism that results in the delivery of toxic effector proteins into neighbouring cells. Virulence is achieved through the targeting of essential components found in either eukaryotic or prokaryotic organisms, including cell wall, membrane lipids, and DNA. Despite the importance of the T6SS as an interbacterial weapon, there still exists an incomplete understanding of how T6SS expressing species can co-exist and the implications of antagonistic responses in shaping polymicrobial communities. Further, these species are often subject to a changing environment and yet, the role of environmental signals and their influence on interspecies interactions and the expression of the T6SS, remain largely unknown. Here, I examined the role of the T6SS in the context of multispecies communities. Using a mix of two antagonistic T6SS strains, Vibrio cholerae V52 and Aeromonas hydrophila SSU, both species co-existed despite active bacterial killing. Fluorescence microscopy analyses revealed survival was possible through the formation of sister-cell clusters. Cluster formation was dependent on T6SS effector delivery, highlighting a unique mechanism where destructive responses can mediate protection within a community. I also examined the environmental signals that activate the T6SS using Pseudomonas aeruginosa PAO1, a strain with a tightly regulated T6SS, as a model system. Results reveal that extracellular DNA, prevalent in cystic fibrosis sputum, activates the H1-T6SS cluster of P. aeruginosa. The addition of excess magnesium ions in the media negates the effect of eDNA on T6SS activation, suggesting eDNA may be a chelator of membrane ions and T6SS activation is a consequence of a perturbed membrane. Overall, I provide new insight into how bacterial communities are shaped, and the type of adaptations bacteria undergo to better survive and compete in the environment.
Open Access
Conserved Interaction between Transferrin and Transferrin-binding Proteins from Porcine Pathogens
(Journal of Biological Chemistry, 2011-06-17) Silva, Leslie P.; Yu, Ronghua; Calmettes, Charles; Yang, Xue; Moraes, Trevor F.; Schryvers, Anthony B.; Schriemer, David C.
Gram-negative porcine pathogens from the Pasteurellaceae family possess a surface receptor complex capable of acquiring iron from porcine transferrin (pTf). This receptor consists of transferrin-binding protein A (TbpA), a transmembrane iron transporter, and TbpB, a surface-exposed lipoprotein. Questions remain as to how the receptor complex engages pTf in such a way that iron is positioned for release, and whether divergent strains present distinct recognition sites on Tf. In this study, the TbpB-pTf interface was mapped using a combination of mass shift analysis and molecular docking simulations, localizing binding uniquely to the pTf C lobe for multiple divergent strains of Actinobacillus plueropneumoniae and suis. The interface was further characterized and validated with site-directed mutagenesis. Although targeting a common lobe, variants differ in preference for the two sublobes comprising the iron coordination site. Sublobes C1 and C2 participate in high affinity binding, but sublobe C1 contributes in a minor fashion to the overall affinity. Further, the TbpB-pTf complex does not release iron independent of other mediators, based on competitive iron binding studies. Together, our findings support a model whereby TbpB efficiently captures and presents iron-loaded pTf to other elements of the uptake pathway, even under low iron conditions.
Open Access
development of a multiplexed protein biomarker assay for colorectal cancer
(2008) Koppel, Jennifer S.; Bathe, Oliver F.; Schriemer, David C.
Open Access
Enabling Structural Proteomics with High Efficiency Protein Enrichment Technology
(2023-09-13) Raval, Shaunak; Schriemer, David C.; MacCallum, Justin L.; Osthoff, Hans D.
The functional state of proteins is inherently flexible, which allows them to interact with other biomolecules, including other proteins, to carry out many of their cellular functions. Understanding the structural dynamics of proteins and their network of associations is key to understanding their role in biology. Proteomics, the collection of mass spectrometry (MS)-based techniques to study proteins, provides a broad view of the organization of protein structure, from an individual dynamic unit to large-scale multiprotein assemblies, enabled by the application of labelling chemistries. This dissertation presents novel analytical workflows and data analysis routines to overcome current challenges in proteomics methods for the identification of protein-protein interactions (PPIs) and the study of protein conformation and dynamics. Affinity purification followed by mass spectrometry (AP-MS) is a prominent approach in the study of PPIs. However, the conventional workflow suffers from low enrichment efficiencies. I present and evaluate a fluidic platform that captures and processes ultralow nanoliter quantities of magnetic particles, simultaneously increasing the efficiency of PPI detection and strongly suppressing non-specific binding. It enables the study of protein conformational analysis directly from cells as I demonstrate first by describing new concepts in data analysis for hydrogen/deuterium exchange mass spectrometry (HX-MS) and second by applying them to proteins isolated directly from cells.
Open Access
Evaluating Chemical Crosslinking as a Tool for Enhancing DNA Damage Repair Interactome Analysis
(2022-03-03) Shariat-Panahi, Ali; Schriemer, David C.; Antonie, Dufour; Cobb, Jennifer
Eukaryotic cells can repair DNA Double-Strand Breaks (DSBs) through a number of mechanisms, including end resection-mediated pathways and non-homologous end joining (NHEJ). An End-Bridging Complex (EBC) comprised of elements from both end resection and NHEJ may be the first complex recruited to DSB sites and play a key role in repair pathway selection. The protein composition of the EBC or how it structurally supports repair pathway choice is not fully understood. Conceptually, Affinity Purification-Mass Spectrometry (AP-MS) methods can be used to determine the composition and subunit stoichiometry of such complexes. However, none of the existing AP-MS workflows combine isolation of complexes with high temporal sampling, both of which are essential for characterizing complexes involved in transient repair processes. In this thesis, I have investigated whether chemical crosslinking incorporated into a DNA-based AP-MS workflow can improve the coverage of DSB repair interactome in S. cerevisiae. To this end, I first evaluated and optimized the RIME protocol for isolating affinity-tagged putative EBC members after DSB-induction. Further, I tested various strategies for incorporating chemical crosslinking into this protocol and increasing protein coverage, using the homo-bifunctional reagent BS3. Lastly, I investigated networks of interaction within the EBC interactome based on the RIME method and evaluated the identified hits for biological significance. My results indicate that the epitope-tagged system I used (hemagglutinin-based) prohibits the incorporation of standard crosslinkers, suggesting that a new strategy is required to take advantage of crosslinking. Nevertheless, the optimized RIME-based AP-MS protocol could successfully isolate known and potentially novel interactors of the EBC using a strategy incorporating multiple baits. Studying the novel interactors identified in these analyses could help in complementing our current knowledge of eukaryotic DSB repair pathway choice and understanding the extent of the DNA damage repair interactome.
Open Access
Function of Oxygen Resistance Proteins in the Anaerobic, Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough
(Journal of Bacteriology, 2003-01) Fournier, Marjorie; Zhang, Yi; Wildschut, Janine D.; Dolla, Alain; Voordouw, Johanna K.; Schriemer, David C.; Vourdouw, Gerrit
Two mutant strains of Desulfovibrio vulgaris Hildenborough lacking either the sod gene for periplasmic superoxide dismutase or the rbr gene for rubrerythrin, a cytoplasmic hydrogen peroxide (H2O2) reductase, were constructed. Their resistance to oxidative stress was compared to that of the wild-type and of a sor mutant lacking the gene for the cytoplasmic superoxide reductase. The sor mutant was more sensitive to exposure to air or to internally or externally generated superoxide than was the sod mutant, which was in turn more sensitive than the wild-type strain. No obvious oxidative stress phenotype was found for the rbr mutant, indicating that H2O2 resistance may also be conferred by two other rbr genes in the D. vulgaris genome. Inhibition of Sod activity by azide and H2O2, but not by cyanide, indicated it to be an iron-containing Sod. The positions of Fe-Sod and Sor were mapped by two-dimensional gel electrophoresis (2DE). A strong decrease of Sor in continuously aerated cells, indicated by 2DE, may be a critical factor in causing cell death of D. vulgaris. Thus, Sor plays a key role in oxygen defense of D. vulgaris under fully aerobic conditions, when superoxide is generated mostly in the cytoplasm. Fe-Sod may be more important under microaerophilic conditions, when the periplasm contains oxygen-sensitive, superoxide-producing targets.
Open Access
Hydra: software for tailored processing of H/D exchange data from MS or tandem MS analyses
(BMC Bioinformatics, 2009-05-27) Slysz, Gordon W.; Baker, Charles A.H.; Bozsa, Benjamin M.; Dang, Anthony; Percy, Andrew J.; Bennett, Melissa; Schriemer, David C.
Background Hydrogen/deuterium exchange mass spectrometry (H/DX-MS) experiments implemented to characterize protein interaction and protein folding generate large quantities of data. Organizing, processing and visualizing data requires an automated solution, particularly when accommodating new tandem mass spectrometry modes for H/DX measurement. We sought to develop software that offers flexibility in defining workflows so as to support exploratory treatments of H/DX-MS data, with a particular focus on the analysis of very large protein systems and the mining of tandem mass spectrometry data. Results We present a software package ("Hydra") that supports both traditional and exploratory treatments of H/DX-MS data. Hydra's software architecture tolerates flexible data analysis procedures by allowing the addition of new algorithms without significant change to the underlying code base. Convenient user interfaces ease the organization of raw data files and input of peptide data. After executing a user-defined workflow, extracted deuterium incorporation values can be visualized in tabular and graphical formats. Hydra also automates the extraction and visualization of deuterium distribution values. Manual validation and assessment of results is aided by an interface that aligns extracted ion chromatograms and mass spectra, while providing a means of rapidly reprocessing the data following manual adjustment. A unique feature of Hydra is the automated processing of tandem mass spectrometry data, demonstrated on a large test data set in which 40,000 deuterium incorporation values were extracted from replicate analysis of approximately 1000 fragment ions in one hour using a typical PC. Conclusion The customizable workflows and user-friendly interfaces of Hydra removes a significant bottleneck in processing and visualizing H/DX-MS data and helps the researcher spend more time executing new experiments and interpreting results. This increased efficiency will encourage the analysis of larger protein systems. The ability to accommodate the tandem MS dimension supports alternative data collection and analysis strategies, as well as higher resolution localization of deuteration where permitted by the fragmentation mechanism.
Open Access
Hydra: software for tailored processing of H/D exchange data from MS or tandem MS analyses
(BMC Bioinformatics, 2009-5-27) Slysz, Gordon W.; Baker, Charles A. H.; Bozsa, Benjamin M.; Dang, Anthony; Percy, Andrew J.; Bennett, Melissa; Schriemer, David C.
Background Hydrogen/deuterium exchange mass spectrometry (H/DX-MS) experiments implemented to characterize protein interaction and protein folding generate large quantities of data. Organizing, processing and visualizing data requires an automated solution, particularly when accommodating new tandem mass spectrometry modes for H/DX measurement. We sought to develop software that offers flexibility in defining workflows so as to support exploratory treatments of H/DX-MS data, with a particular focus on the analysis of very large protein systems and the mining of tandem mass spectrometry data. Results We present a software package ("Hydra") that supports both traditional and exploratory treatments of H/DX-MS data. Hydra's software architecture tolerates flexible data analysis procedures by allowing the addition of new algorithms without significant change to the underlying code base. Convenient user interfaces ease the organization of raw data files and input of peptide data. After executing a user-defined workflow, extracted deuterium incorporation values can be visualized in tabular and graphical formats. Hydra also automates the extraction and visualization of deuterium distribution values. Manual validation and assessment of results is aided by an interface that aligns extracted ion chromatograms and mass spectra, while providing a means of rapidly reprocessing the data following manual adjustment. A unique feature of Hydra is the automated processing of tandem mass spectrometry data, demonstrated on a large test data set in which 40,000 deuterium incorporation values were extracted from replicate analysis of approximately 1000 fragment ions in one hour using a typical PC. Conclusion The customizable workflows and user-friendly interfaces of Hydra removes a significant bottleneck in processing and visualizing H/DX-MS data and helps the researcher spend more time executing new experiments and interpreting results. This increased efficiency will encourage the analysis of larger protein systems. The ability to accommodate the tandem MS dimension supports alternative data collection and analysis strategies, as well as higher resolution localization of deuteration where permitted by the fragmentation mechanism.
Open Access
Identification and Functional Characterization of Protein Kinase A-catalyzed Phosphorylation of Potassium Channel Kv1.2 at Serine 449
(Journal of Biological Chemistry, 2009-04-22) Johnson, Rosalyn P.; El-Yazbi, Ahmed F.; Hughes, Morgan F.; Schriemer, David C.; Walsh, Emma J.; Walsh, Michael P.; Cole, William C.
Vascular smooth muscle Kv1 delayed rectifier K+ channels (KDR) containing Kv1.2 control membrane potential and thereby regulate contractility. Vasodilatory agonists acting via protein kinase A (PKA) enhance vascule smooth muscle Kv1 activity, but the molecular basis of this regulation is uncertain. We characterized the role of a C-terminal phosphorylation site, Ser-449, in Kv1.2 expressed in HEK 293 cells by biochemical and electrophysiological methods. We found that 1) in vitro phosphorylation of Kv1.2 occurred exclusively at serine residues, 2) one major phosphopeptide that co-migrated with 449pSASTISK was generated by proteolysis of in vitro phosphorylated Kv1.2, 3) the peptide 445KKSRSASTISK exhibited stoichiometric phosphorylation by PKA in vitro, 4) matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy (MS) and MS/MS confirmed in vitro Ser-449 phosphorylation by PKA, 5) in situ phosphorylation at Ser-449 was detected in HEK 293 cells by MALDI-TOF MS followed by MS/MS. MIDAS (multiple reaction monitoring-initiated detection and sequencing) analysis revealed additional phosphorylated residues, Ser-440 and Ser-441, 6) in vitro 32P incorporation was significantly reduced in Kv1.2-S449A, Kv1.2-S449D, and Kv1.2-S440A/S441A/S449A mutant channels, but Kv1.2-S440A/S441A was identical to wild-type Kv1.2 (Kv1.2-WT), and 7) bath applied 8-Br-cAMP or dialysis with PKA catalytic subunit (cPKA) increased Kv1.2-WT but not Kv1.2-S449A current amplitude. cPKA increased Kv1.2-WT current in inside-out patches. Rp-CPT-cAMPS reduced Kv1.2-WT current, blocked the increase due to 8-Br-cAMP, but had no effect on Kv1.2-S449A. cPKA increased current due to double mutant Kv1.2-S440A/S441A but had no effect on Kv1.2-S449D or Kv1.2-S440A/S441A/S449A. We conclude that Ser-449 in Kv1.2 is a site of PKA phosphorylation and a potential molecular mechanism for Kv1-containing KDR channel modulation by agonists via PKA activation.
Open Access
Identification of dipeptidase-1 as an organ-selective adhesion receptor utilized by neutrophils and metastatic cancer cells in the liver and lungs
(2018-04-30) Roy Choudhury, Saurav; Senger, Donna L.; Kubes, Paul; Liao, Shan; Morris, Don G.; Schriemer, David C.; Ferri, Lorenzo Edwin
Lungs and liver are two major sites of neutrophil trafficking and inflammatory disease. Neutrophil recruitment in response to an inflammatory cue is a sequentially coordinated process where adhesion molecules expressed on the endothelium of a given organ mediate different steps in the classical leukocyte recruitment cascade [1]. However, molecules identified as being central in the canonical schema of neutrophil recruitment to different organs (mesentery, skin, and cremaster muscle) are not required in the inflamed pulmonary and hepatic vasculatures [2-8]. Using an unbiased functional screen in vivo, we isolated a peptide-displaying phage that homed to the liver and lungs of mice treated with a bacterial inflammatory stimulus (lipopolysaccharide). Employing intravital microscopy, we found that this phage, or its corresponding displayed-peptide, termed LSALT herein, inhibited the adhesion of neutrophils in the inflamed lungs and liver vasculatures in response to LPS. The corresponding synthetic peptide also reduced the metastatic colonization of melanoma cells to the lungs in human xenograft and immunocompetent mouse models. Using biochemical, genetic and confocal intravital imaging approaches we identified dipeptidase-1 (DPEP1) as the functional target of this peptide and established its role as a physical adhesion receptor for neutrophil and metastatic cancer cell adhesion independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment and cancer metastasis to the lungs and liver, and in models of Acute Respiratory Distress Syndrome (ARDS), prevented septic lung injury and mortality. This study identified DPEP1 as an organ-selective vascular endothelial adhesion receptor for the recruitment of neutrophils and metastatic cancer cells to the lungs and liver and identifies DPEP1 as a novel therapeutic target for systemic inflammatory disorders as well as organ-selective metastatic diseases.
Open Access
Incorporation of Chiral Selectors into a Water Stationary Phase for use in Supercritical Fluid Chromatography
(2018-06-14) Frantz, Jackson Joseph; Thurbide, Kevin B.; Shi, Yujun J.; Gailer, Jürgen G.; Schriemer, David C.
The direct separation of enantiomers in column chromatography is an important field that has seen considerable maturation and explosive growth in the last three decades. This development has been spurred by needs for fast and cost-effective analysis in application areas such as pharmaceuticals, agrochemicals, and environmental toxicology. Despite this progress, method development in chiral analysis is often difficult and prohibitively costly due to expensive columns and lack of universally applicable selectors. This thesis presents a new method for chiral separations using a modified aqueous stationary phase. By incorporating chiral selectors in a liquid phase on stainless steel capillary tubing, chiral separations may be achieved conveniently and at extremely low cost. Further, the application of unmodified carbon dioxide as a mobile phase for these separations eliminates the need for costly and potentially harmful organic solvents in routine operation, and also allows the use of the universal flame ionization detector. Toward this end, efforts to effect separations using native and derivatized beta cyclodextrin selectors were undertaken, with some promising results for the separation of some flavanone enantiomers. Especially notable was the extremely potent selectivity obtained at novel operating conditions such as subambient temperatures. The thermodynamic and kinetic performance of the system was evaluated revealing some highly novel characteristics. To enable separation of a greater diversity of chiral analytes, the macrocyclic antibiotic selector vancomycin was also investigated. Excellent performance was obtained using this selector for the separation of some phenoxypropionic acid enantiomers. The addition of additives was highly relevant to these separations. Most notably the improvement with ternary agents such as triethylamine revealed the utility of the water stationary phase as a unique molecular scaffolding for promoting chiral discrimination. Finally, applications demonstrating the separation of both a flavanone and phenoxypropionic acid in complex sample matrices, as well as mixed selector phases utilizing both beta cyclodextrin and vancomycin for the simultaneous separation of multiple chiral pairs highlighted the versatility of the novel separation system.
Open Access
Integration of deep transcriptome and proteome analyses reveals the components of alkaloid metabolism in opium poppy cell cultures
(BMC Plant Biology, 2010-11-18) Desgagné-Penix, Isabel; Khan, Morgan F.; Schriemer, David C.; Cram, Dustin; Nowak, Jacek; Facchini, Peter J.
Papaver somniferum (opium poppy) is the source for several pharmaceutical benzylisoquinoline alkaloids including morphine, the codeine and sanguinarine. In response to treatment with a fungal elicitor, the biosynthesis and accumulation of sanguinarine is induced along with other plant defense responses in opium poppy cell cultures. The transcriptional induction of alkaloid metabolism in cultured cells provides an opportunity to identify components of this process via the integration of deep transcriptome and proteome databases generated using next-generation technologies.
Open Access
Integrative Structural Model of DNA-PKcs in the Initial Steps of Non-Homologous End Joining
(2020-06-04) Hepburn, Morgan Rose; Schriemer, David C.; Lees-Miller, Susan P.; Ng, Kenneth Kai Sing; Williams, Gareth J.; Huang, Lan
Non-homologous end joining (NHEJ) performs untemplated repair of DNA double strand breaks (DSBs). Despite lack of a template, intricate repair, coordinated by the core NHEJ factors, can repair breaks with minimal to no alterations. Initiating repair, Ku70/80 binds to the free DNA ends, and interacts with the large protein kinase, DNA dependent protein kinase catalytic subunit (DNA-PKcs), forming the holoenzyme DNA-PK. Holoenzymes can synapse across the break to tether the DNA ends. Assembly of the initial synaptic complex and its role in NHEJ is poorly understood, as final ligation requires a structural rearrangement of this initial complex. To better understand DNA-PKcs’ role in NHEJ, an integrative structural model of DNA-PKcs in the initial stages of NHEJ was developed using mass spectrometry (MS) techniques. Due to technical challenges working with DNA-PKcs, each of the MS techniques were optimized for the system. Hydrogen deuterium exchange (HX) methods were optimized on a nano-spray HX system, allowing for differential HX analysis of bead bound DNA-PKcs complexes with high sequence coverage, and 5X improvement in protein consumption. Reversible crosslinking and peptide fingerprinting (RCAP) was optimized to allow for direct detection of DNA binding peptides, using a single sample. Finally, given the benefits of DNA-PKcs complex assembly on beads to limit heterogeneity, an on-bead crosslinking method was developed. Mass Spec Studio was used to accurately identify many crosslinks, which can be utilized for a label free quantitation comparison of states. Using HX-MS to explore DNA-PKcs conformational changes from binding to activation of the kinase, an allosteric pathway was identified in DNA-PKcs connecting DNA-binding with the kinase domain. Nucleotide loading of the kinase domain revealed that DNA-PK occupies a tensed state when active. From integrative structural modelling, with the XL-MS restraints, a model with a precision of 13.5Å was reported, revealing a symmetric DNA-PK dimer, with head-to-head interactions. In our synaptic model, the DNA ends are positioned with a large offset, protected by a previously uncharacterized plug domain of DNA-PKcs. We propose the initial formation of the synaptic complex allows for a hierarchical processing of DNA ends and assembly of a core NHEJ scaffold.
Open Access
Investigating the binding interaction between granulocyte-macrophage colony-stimulating factor and the soluble alpha subunit of its receptor through hydrogen deuterium exchange mass spectrometry
(2006) Vande Graaf, Jaclyn Louise; Brown, Christopher B.; Schriemer, David C.
Open Access
Lactoferrin binding protein B - a bi-functional bacterial receptor protein
(PLOS Pathogens, 2017-3-3) Ostan, Nicholas K. H.; Yu, Rong-Hua; Ng, Dixon; Lai, Christine Chieh-Lin; Pogoutse, Anastassia K.; Sharpe, Vladimir; Hepburn, Morgan; Sheff, Joey; Raval, Shaunak; Schriemer, David C.; Moraes, Trevor F.; Schryvers, Anthony B.
Lactoferrin binding protein B (LbpB) is a bi-lobed outer membrane-bound lipoprotein that comprises part of the lactoferrin (Lf) receptor complex in Neisseria meningitidis and other Gram-negative pathogens. Recent studies have demonstrated that LbpB plays a role in protecting the bacteria from cationic antimicrobial peptides due to large regions rich in anionic residues in the C-terminal lobe. Relative to its homolog, transferrin-binding protein B (TbpB), there currently is little evidence for its role in iron acquisition and relatively little structural and biophysical information on its interaction with Lf. In this study, a combination of crosslinking and deuterium exchange coupled to mass spectrometry, information-driven computational docking, bio-layer interferometry, and site-directed mutagenesis was used to probe LbpB:hLf complexes. The formation of a 1:1 complex of iron-loaded Lf and LbpB involves an interaction between the Lf C-lobe and LbpB N-lobe, comparable to TbpB, consistent with a potential role in iron acquisition. The Lf N-lobe is also capable of binding to negatively charged regions of the LbpB C-lobe and possibly other sites such that a variety of higher order complexes are formed. Our results are consistent with LbpB serving dual roles focused primarily on iron acquisition when exposed to limited levels of iron-loaded Lf on the mucosal surface and effectively binding apo Lf when exposed to high levels at sites of inflammation.