2009 Anachem/SAS Symposium
An on-line registration form is available
here |
The 2009 Anachem Symposium will be held on Thursdayday, October 29th at the
Burton Manor in Livonia.see map The preliminary
program is listed below. More information about the meeting will be posted
here as it becomes available Meeting registration includes the meeting
sessions, instrument exhibit, workshops, lunch and a post-meeting reception.
You do not have to be an Anachem Member to attend the
Anyone wishing to present a poster should contact Patsy Coleman at
pcoleman@chem.wayne.edu. The deadline for submission of titles and abstracts
(up to a 250 words) is due Oct 3, 2008.
Don't forget to check for late changes in the
program.
MORNING SESSION
8:30 AM> Characterization of the Binders in the Rock Art of Cueva La
Conga, Nicaragua. 8:50 AM> A New AP-MALDI Ion Source for Producing Singly or Highly-Charged Molecular Ions at Will,. Ellen D. Inutan1, Thushani N. Herath1, S. Alexandru Cernat1, James Wager-Miller2, Ken Mackie2, J. Michael Walker2, Charles N. McEwen3, Sarah Trimpin1*, Wayne State University, Detroit, MI 9:10 AM> Development and Characterization of an Optimized Sample Preparation Procedure for the Metabolomic Analysis of ß-Cells by Liquid Chromatography - Time of Flight Mass Spectrometry, Matthew A. Lorenz and Robert T. Kennedy, University of Michigan, Ann Arbor, MI 9:30 AM> Bulk and Single Molecule Analysis of the Effects of 2' Modifications on Molecular Beacons, Sharla L. Wood and David Rueda, Wayne State University, Detroit, MI 9:50 AM> Effect of an Intermittent Administration of MnCl2 on the Dopaminergic System, Rabab A. Aoun, Madiha Khalid, Tiffany A. Mathews, Wayne State University, Detroit, MI 10:10 AM> Brain-derived Neurotrophic Factor Deficient Mice Appear to be Hyperdopaminergic, K. E. Bosse*, F. Maina, M. M. France*, J. J. P. Roberts and T. A. Mathews, Wayne State University, Detroit, MI
ANACHEM AWARD SESSION ROOM
3 8:30 AM> Probing the Effects of Cationization on the Structure and Stability of Nucleic Acids via IRMPD Action Spectroscopy and Theory, Mary T. Rodgers,1,* Yuan-wei Nei,1 Ranran Wu,1 B. Scott Fales,1 Nathaniel O. Fujamade,1, Nuwan Hallowita,1 Jos Oomens,2 and Jeffrey D. Steill2, 1Wayne State University, Detroit, MI, USA, 2FOM Institute for Plasma Physics "Rijnhuizen", Nieuwegein, The Netherlands 9:00 AM> FT-ICR Mass Spectrometric Characterization of Curacin A Biosynthesis, Kristina Håkansson, Bo Wang, Haichuan Liu, Liangcai Gu, and David H. Sherman, University of Michigan, Ann Arbor, MI 9:30 AM> Innovations in Bioanalytical Mass Spectrometry: An Affinity Reagent Free Approach to Protein Quantitation, Michael J. Ford, Richard C. Jones; Ravi Amunugama, Ruth Vanbogelen and Michael Pisano, NextGen Sciences Inc., Ann Arbor, MI 10:00 AM>Determining Protein Quaternary Structures using Constraints from Gas-phase Measurements, Brandon Ruotolo, University of Michigan, Ann Arbor, MI
Abstract
Who Should Attend
Biography
PLENARY LECTURE Dr. McLuckey's research emphases have been placed in the areas of gas-phase ion chemistry and instrumentation for organic and biological mass spectrometry. Fundamental aspects of ionization, unimolecular reactions, and bi-molecular reactions have been studied with the goal of improving the capabilities of analytical mass spectrometry. Ion activation, ion/molecule reactions, and ion/ion reactions have been major focal areas within the context of the mass spectrometry/mass spectrometry experiment. Instrumentation for tandem mass spectrometry has also been highlighted with emphasis on electrodynamic ion traps and ion trap/hybrid instruments. This research has been described in over 255 papers appearing in the peer-reviewed literature. He has been awarded six U.S. Patents on various technologies associated with mass spectrometry. The major current areas of emphasis are the identification and characterization of macro-molecules, primarily via whole molecule tandem mass spectrometry, and ion/ion reaction chemistry. Recognition for the work has included the Biemann Medal from the American Society for Mass Spectrometry in 1997, Oak Ridge National Laboratory Scientist of the Year in 1999, The Lockheed Martin Nova Award in 1999, The Curt Brunneé Prize from the International Mass Spectrometry Society in 2000, and the American Chemical Society Division of Analytical Chemistry Chemical Instrumentation Award in 2007. Dr. McLuckey has served as an editor of the International Journal of Mass Spectrometry since 1977 and either serves or has served on the editorial boards of Analytical Chemistry, the Journal of Mass Spectrometry, Rapid Communications in Mass Spectrometry, the Journal of the American Society for Mass Spectrometry, Mass Spectrometry Reviews, and the Chinese Journal of Mass Spectrometry. He has co-taught short courses at the annual American Society for Mass Spectrometry on Fundamental Aspects of Mass Spectrometry (3 years) and Quadrupole Ion Traps (6 years). He was recently elected Vice President for Programs and President-Elect of the American Society for Mass Spectrometry.
12:30PM - 1:30PM
AFTERNOON SESSION 1:30 PM> Structures and Binding Energies of Noncovalent Complexes of Peptidomimetic Protonated Nitrogen Bases with 18-Crown-6, Yu Chen, M.T. Rogers, Wayne State University, Detroit, MI 1:50 PM> Atmospheric Pressure Femtosecond Laser Imaging Mass Spectrometry, Yves Coello, A. Daniel Jones, Tissa C. Gunaratne and Marcos Dantus, Michigan State University, East Lansing, Michigan 2:10 PM> Probing the Effects of Protonation on the Structure and Stability of Uracil, Thiouracils, and Methylthiouracils via IRMPD Action Ion Spectroscopy and Theoretical Calculation, Yuan-wei Nei1 , Tolulope E. Akinyemi1, Jeffrey D. Steill2 , Jos Oomens2 , and M. T. Rodgers1* 1Wayne State University, Detroit, MI, 2FOM Institute for Plasma Physics "Rijnhuizen", Nieuwegein, The Netherlands 2:30 PM> A Sulfonium Ion Containing Protein Modification Reagent and Tandem Mass Spectrometry for the Study of Protein Surface Accessibility and Protein Interactions, Xiao Zhou and Gavin E. Reid, Michigan State University, East Lansing, MI 2:50 PM> ESI-MS Detection of Segmented Flow and Application to Fraction Collection from Capillary Liquid Chromatography, Qiang Li1, Jian Pei1, Mike S. Lee2, Gary A. Valaskovic3 and Robert T. Kennedy1, 1. University of Michigan, Ann Arbor, MI, 2. Milestone Dev Serv Newtown, PA , 3. New Object Inc, Cambridge, MA 3:10 PM> Spray Desorption Collection, Afrand Kamali, Andre Venter, Western Michigan University, Kalamazoo, MI
Abstract
Who should attend
Biography
AFTERNOON WORKSHOP 2 ROOM 2
Abstract
Who Should Attend
Biography
Spray Desorption Collection (SDC) - A Novel Sampling Technique Coupled with UV Absorbance Spectrometer for Nano Particle Analysis, Shashank Jain, Andre Venter, Western Michigan University, Kalamazoo, MI Cation Aza-crown Complexes: Determination of Structures and Bond Dissociation Energies Using Guided Ion Beam Tandem Mass Spectrometry and Quantum Chemical Calculations, Calvin Austin and M.T. Rodgers, Wayne State University, Detroit, MI Accumulation of Manganese in Kidney, Liver, and Brain upon Intermittent Subcutaneous Injections of MnCl2, Rabab A. Aoun, Madiha Khalid, Tiffany A. Mathews, Wayne State University, Detroit MI Determining the Efficacy of Non-Destructive Pre-Treatment Methods on Known Age Textile Samples by Gas Chromatography-Mass Spectrometry Analysis, Deidre Hardemon and Ruth Ann Armitage, Eastern Michigan University, Ypsilanti, MI Alterations in Dopamine Neurotransmitter Dynamics in Brain-derived Neurotrophic Factor Deficient Mice, K. E. Bosse, M. M. France*, J. J. P. Roberts and T. A. Mathews, Wayne State University, Detroit, MI Qualitative and Quantitative Analyses of Lipidic Binders in Rock Paintings, Christina Phillips and Ruth Ann Armitage, Eastern Michigan University, Ypsilanti, MI Gas-Phase Conformations of Cationized Mononucleotides Determined by Infrared Multiphoton Dissociation Spectroscopy and Theoretical Modeling, Ranran Wu and M. T. Rodgers*, Wayne State University, Detroit, MI Pharmaceutical Cleaning Validation by Spray Desorption Collection, Kevin Douglas, Shashank Jain, Andre Venter, Western Michigan University, Kalamazoo,MI Fast Analysis of GABA and Glutamate in Dialysis Samples Using a Monolithic Column , Stella Wisidagama and Tiffany A. Mathews, Wayne State University, Detroit, MI
ABSTRACTS
ANACHEM AWARD SESSION Probing the Effects of Cationization on the Structure and Stability of Nucleic Acids via IRMPD Action Spectroscopy and Theory, Mary T. Rodgers,1,* Yuan-wei Nei,1 Ranran Wu,1 B. Scott Fales,1 Nathaniel O. Fujamade,1 Nuwan Hallowita,1 Jos Oomens,2 and Jeffrey D. Steill2, 1Department of Chemistry, Wayne State University, Detroit, MI, USA, 2FOM Institute for Plasma Physics "Rijnhuizen", Nieuwegein, The Netherlands Metal cations are known to influence the structures and mediate the functions of nucleic acids. Metal cation-nucleic acid interactions are also used as tools for molecular biology studies. Furthermore, the binding of metal cations to the phosphate backbone of nucleic acids can lead to cleavage of the phosphodiester linkages, and in some cases, selective cleavage of the 3'-phosphate ester linkage(s). Thus, the binding of an appropriate metal cation to a nucleic acid may potentially be used to selectively cleave phosphate ester linkages and result in facile sequencing. However, the current understanding of the structural and functional roles that metal cation-nucleic acids interactions play is rather limited, largely because only limited information regarding the influence of metal cation binding on the structure and stability of nucleic acids is currently available. To better understand how cation binding influences the structure and stability of nucleic acids, we have undertaken systematic studies aimed at the characterization of the structures, cation binding affinities, and activation propensities of a wide variety of model and biologically relevant deprotonated, protonated, and metal cationized phosphate esters. In nucleic acids, the phosphate groups along the backbone are coupled to the adjacent ribose moieties via a 1° alkyl phosphate linkage (5'-phosphate ester linkage) and a 2° alkyl phosphate linkage (3'-phosphate ester linkage). Thus, diethyl phosphate serves as an excellent yet simple model for nucleic acids, possessing both 1° alkyl phosphate ester linkages and a single highly acidic hydrogen atom. Five phosphate esters are examined in the present study, diethyl phosphate (DEP) and the four 2'-deoxymononucleotide-5'-phosphate esters (dA5'p, dC5'p, dG5'p, and dT5'p). Electrospray ionization (ESI) is employed to generate the deprotonated, protonated, and metal cationized (Na+, Mg2+) complexes of these species. These complexes were isolated and subjected to infrared multiphoton dissociation (IRMPD) in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS). A free electron laser (FEL) whose output frequency is scanned from approximately 600 to 1800 cm-1 is used for IRMPD excitation. Because dissociation of the reactant complexes will only occur if the complex absorbs at the excitation wavelength, the resultant IRMPD action spectrum provides the IR spectrum of the reactant complex over the frequency range scanned. The observed IRMPD products also provide insight into the nature of the binding and the stability of the phosphate ester. Density functional theory calculations at the B3LYP/6-31G* level of theory were performed to determine the structures/binding modes and linear IR spectra of the various low-energy conformations of the species studied experimentally. 300 cycles of simulated annealing (1000 K) using the AMBER force field were performed to determine candidate structures for higher level optimization for each species examined theoretically. Typically, 25-35 of the most stable structures found for each species from the annealing process were examined using density functional theory. The relative stabilities (including zero point energy corrections) of the various low-energy conformers of each species are computed using an extended basis set, B3LYP/6-311+G(2d,2p). The observed dissociation pathways clearly establish the phosphate group as the most favorable site for deprotonation and cationization. In contrast, protonation occurs preferentially at the nucleobase except in DEP and dT5'p. Additional stabilization is generally achieved via hydrogen bonding and additional chelation interactions with the metal cation(s). The IRMPD action spectra of these phosphate esters exhibit spectral signatures for the phosphate group lie in the range between 650 and 1400 cm-1, while those of the nucleobase lie between 1400 and 1750 cm-1. Protonation and cationization clearly influence the IR spectra in the fingerprint regions of both the phosphate group and the nucleobase for all species investigated. The observed IRMPD action spectra suggest that the cations interact with both the phosphate group and the nucleobase (and in some cases with the sugar as well) in agreement with the theoretical calculations. Differences in the structure of these species as a function of the charge state, cation, and identity of the nucleobase are examined in detail. FT-ICR Mass Spectrometric Characterization of Curacin A Biosynthesis, Kristina Håkansson, Bo Wang, Haichuan Liu, Liangcai Gu, and David H. Sherman, Department of Chemistry and Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA Nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are large, modular, multifunctional enzyme complexes that catalyze the formation of diverse natural products. Each NRPS/PKS module catalyzes a specific elongation, redox, or cyclization step in the biosynthesis. These enzymes represent a group of biocatalysts that can be engineered with the goal of fermentative production of novel antibiotics and anticancer agents. However, for a targeted approach, it is necessary to understand the detailed catalytic mechanisms. The high mass accuracy and resolution of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) render it an excellent tool to probe covalent intermediates in NRPS and PKS biosynthesis. Curacin A [1] is a mixed PKS/NRPS marine cyanobacterial metabolite with anticancer activity. The structure of curacin A contains several intriguing features, including a cyclopropane ring and a terminal double bond. Here, we will discuss both top-down and bottom-up approaches to interrogate curacin A biosynthesis. We have characterized a range of excised functional folds from the curacin A biosynthetic pathway. Top-down FT-ICR MS revealed that a pair of crotonase superfamily members catalyze dehydration and decarboxylation of an acyl carrier protein (ACP)-bound intermediate [2]. In addition, top-down and bottom-up FT-ICR MS were used to reveal the mechanism of the chain initiation module of the curacin A PKS. A domain from the GCN5-related N-acetyltransferase (GNAT) superfamily catalyzes decarboxylation of malonyl-CoA, followed by a direct S-acetyltransfer to an adjacent ACP [3]. This bifunctional decarboxylase/S-acetyl transferase activity is unprecedented for the GNAT superfamily. In further work, we showed that a curacin A enoylreductase (ER) domain catalyzes an unprecedented cyclopropanation reaction of a chlorinated intermediate [4]. Chlorination occurs via an upstream halogenase domain. Finally, we have shown that the terminal double bond in curacin A is introduced through a decarboxylative reaction by the terminal thioesterase [5]. This reaction occurs on a sulfated intermediate generated by an adjacent upstream sulfotransferase. 1. Chang, Z. X.; Sitachitta, N.; Rossi, J. V.; Roberts, M. A.; Flatt, P. M.; Jia, J. Y.; Sherman, D. H.; Gerwick, W. H. J. Nat. Prod. 2004, 67, 1356-1367.,2. Gu, L.; Jia, J.; Liu, H. C.; Håkansson, K.; Gerwick, W. H.; Sherman, D. H. J. Am. Chem. Soc. 2006, 128, 9014-9015.,3. Gu, L.; Geders, T. W.; Wang, B.; Gerwick, W. H.; Håkansson, K.; Smith, J. L.; Sherman, D. H. Science 2007, 318, 970-974.,4. Gu, L.; Wang, B.; Kulkarni, A.; Geders, T. W.; Grindberg, R. V.; Gerwick, L. G.; Håkansson, K.; Wipf, P.; Smith, J. L.; Gerwick, W. H.; Sherman, D. H. Nature 2009, 459, 731-735..5. Gu, L.; Wang, B.; Kulkarni, A.; Gehret, J. J.; Lloyd, K.; Gerwick, L.; Gerwick, W. H.; Wipf, P.; Håkansson, K.; Smith, J. L.; Sherman, D. H. J. Am. Chem. Soc. 2009, accepted. Innovations in Bioanalytical Mass Spectrometry: An Affinity Reagent Free Approach to Protein Quantitation, Michael J. Ford; Richard C. Jones; Ravi Amunugama, Ruth Vanbogelen and Michael Pisano, NextGen Sciences Inc., Ann Arbor, MI In the post-genome era, the use of molecular biomarkers is becoming de rigueur for programs in research and in drug development. The biomarker testing market has a proven record of revenue generation ($612 MM in 2007) and is estimated to have an annual growth rate of 23.5% based on currently available biomarker assays. Proteomic technologies have been used successfully for biomarker discovery projects producing lists of many candidate protein biomarkers; when integrated with genomic data and literature mining there can be hundreds of candidates for a given study. However, further verification work is typically limited by the small number of proteins for which there are commercially available assays (~500 human proteins). If researchers opt to develop assays for these candidates using traditional antibody-based approaches, the cost would likely be over $25K/protein and the timeline would be a couple of years or more. Thus, for protein biomarkers, assay development is a current bottleneck. In this presentation a Liquid Chromatography/Mass Spectrometry (LC/MS) based approach for the validation of protein biomarkers will be discussed. Quantitation is achieved using surrogate peptides generated from an enzymatic digest of the native protein in a biological sample. The application of LC-Selected Reaction Monitoring mass spectrometry (LC-SRM/MS) technology enables the quantitation of the surrogate peptide in the digested biological sample. The stoichometric relationship between the peptide and the native protein can be used to infer the protein level in a given sample. Ultimately the use of an isotope labeled internal standard peptide yields absolute quantitation data. The primary objective of this workflow is to significantly decrease the cost and timelines for assay development and biomarker validation. Determining Protein Quaternary Structures using Constraints from Gas-phase Measurements, Brandon Ruotolo. University of Michigan, Ann Arbor, MI Structural biology is ultimately concerned with determining high-resolution structures of all the functional macromolecules within living cells and tissues. While high-detail structural information can be obtained by X-ray diffraction analysis, this experiment requires the availability of a sufficient quantity of homogenous material and definition of suitable crystallization parameters. Both conditions are often difficult to meet and the number of structures of multi-subunit complexes deposited in structural databases remains relatively low. Alternative methodologies such as electron microscopy (EM) and small angle X-ray scattering (SAXS) allow the determination of the surface envelope of complexes of sufficient dimensions but interpretation of these data is aided by detailed knowledge of complex composition, and is limited to homogeneous complexes. Consequently there is a need to apply new approaches that define the subunit stoichiometry, composition, and shape of heterogeneous macromolecular complexes of biological importance. Over the past several years, we have been developing ion mobility-mass spectrometry (IM-MS) methods for the analysis of large protein assemblies. IM separates ions based on their ability to traverse a chamber filled with inert neutral molecules under the influence of a weak electric field. Ions that are large undergo a greater number of collisions with neutral molecules and thus take more time to elute from the chamber than smaller, more compact molecules. Ion size is, therefore, the primary information content of IM separation and computational approaches can be used in conjunction with this information and MS data to assign the overall topology and structure to the assembly. This presentation will focus a number of heteromeric and homomeric protein complexes currently under investigation to illustrate this approach.
Advances in Academic Research Characterization of the Binders in the Rock Art of Cueva La Conga, Nicaragua,Ran Li and Ruth Ann Armitage, Department of Chemistry, Eastern Michigan University, Ypsilanti, MI Cueva La Conga is the only recorded painted cave in Nicaragua, in a part of that country about which little is known archaeologically. The rock art, which includes carved rock formations as well as paintings and handprints, may provide clues about the past people and culture of this area. Determining the age of the paintings will help to understand the possible cultural relationships between known cultures and the rock art of Cueva La Conga. None of the rock art of Nicaragua has been dated before. Radiocarbon dates for the charcoal images range back over 1000 years. To date the red, yellow, and purple images requires that an organic binder was used and remains to be extracted and dated using accelerator mass spectrometry to determine the 14C content. We are using thermally assisted hydrolysis/methylation-gas chromatography-mass spectrometry (THM-GC-MS) to study the composition of the paints to determine if any binder material remains to be dated, and to compare that composition to known binder materials. Comparing the compositions of the paint and unpainted limestone will allow us to determine if a reliable date is likely to be obtained. This is the first comprehensive study where the chemical characterization of the paint was considered in the sampling of the rock art to be dated. We will describe the inherent difficulties of reconciling good analyses with preservation of these irreplaceable and at-risk cultural materials. A New AP-MALDI Ion Source for Producing Singly or Highly-Charged Molecular Ions at Will, Ellen D. Inutan1, Thushani N. Herath1, S. Alexandru Cernat1, James Wager-Miller2, Ken Mackie2, J. Michael Walker2, Charles N. McEwen3, Sarah Trimpin1*, Wayne State University, Detroit, MI A sensitive new atmospheric pressure (AP) matrix-assisted laser desorption/ionization (MALDI) source compatible with commonly available mass spectrometers provides either highly charged ions (e.g. +13 for lysozyme), as observed in electrospray ionization (ESI), or singly-charged ions as typically observed in MALDI simply by choice of matrix or matrix preparation conditions. This new transmission geometry (TG) AP-MALDI source operates entirely free of any electric fields to improve tissue imaging, and to eliminate ion rim losses at the atmospheric/vacuum interface while AP condition aids rapid cooling of the ions. In the configuration we use, the AP-MALDI source was interfaced to a standard AP ion source common on liquid chromatography mass spectrometry (MS) instruments (Thermo Scientific Orbitrap Exactive, Waters LCT Premier). The focused nitrogen laser beam (337 nm; 300 µJ/pulse) is guided through a glass or quartz sample holder, positioned closely (1 mm) in front of the mass spectrometer ion entrance aperture allowing enhanced ion sampling by the combination of the forward momentum of the laser generated plume and flow entrapment into the gas diffusing from the AP to vacuum region through the MS orifice. This source design has significant potential analytical utility, e.g. tissue imaging at AP conditions along with rapid solvent-free matrix application for undisturbed localization of tissue molecules. The production of highly charged ions fill a significant gap in our understanding of the MALDI process, and likely ESI. A second distinct ionization mechanism produced only singly charged MALDI ions simply by choosing the matrix and preparation conditions. The freedom of charge state selection is unprecedented with any ionization method and has considerable practical analytical utility because highly charged ions extend the mass range of mass limited instruments, improve the detection efficiency (sensitivities of 5 attomoles for angiotensin has been recorded), and facilitates structural characterization through fragment analysis while singly charged ions reduce mass spectral complexity in mixture analysis. Development and Characterization of an Optimized Sample Preparation Procedure for the Metabolomic Analysis of ß-Cells by Liquid Chromatography - Time of Flight Mass Spectrometry, Matthew A. Lorenz and Robert T. Kennedy, University of Michigan, Ann Arbor, MI Despite considerable effort from researchers, the metabolic pathways associated with glucose stimulated insulin secretion (GSIS) in ß-cells are not fully understood. In addition, sample preparation procedures crucial for accurate metabolomic analysis of cultured mammalian cells such as the clonal ß-cell line, INS-1, valuable in the study of GSIS have yet to be sufficiently characterized and optimized for LC-MS based metabolomic analysis. To these ends, we have developed and optimized an extraction procedure for adherent mammalian cells well suited to global LC-MS based metabolomic studies and applied this method to the directed and undirected metabolomic analysis of GSIS in ß-cells. This simplified sample preparation procedure involves quenching of metabolism using direct contact with liquid nitrogen followed by a single extraction step with a methanol-water solvent system. The procedure was optimized for extraction solvent, volume, time, and number of extraction steps. The benefits and acceptability of rinsing cells with water to remove extracellular metabolites and buffers detrimental to LC-MS sensitivity are also assessed. This method was applied to INS-1 cells subjected to glucose stimulation to elucidate metabolites and pathways associated with GSIS. Extracts were characterized using both directed and undirected approaches employing HILIC with TOF-MS to resolve, detect, and identify as many metabolic species as possible. Multiple chromatographic features were observed to change under glucose stimulation and characterized by accurate mass determination comparison to authentic standards. Bulk and Single Molecule Analysis of the Effects of 2' Modifications on Molecular Beacons, Sharla L. Wood and David Rueda*. Wayne State University, Detroit, MI In this study, we have used fluorescence resonance energy transfer (FRET) and single molecule spectroscopy to analyze the effects that 2' backbone modifications have on the dynamics of molecular beacons (MB). Molecular beacons are advantageous for live cell assays because they only fluoresce in the presence of the target molecule, negating the need to wash away unbound probes, which allows delivery into living cells. Molecular beacons with 2' backbone modifications are expected to prevent nuclease degradation in the cells. 2'F, 2'OMe, and 2'H modifications were characterized and compared to the parent 2'OH MB via steady state, time resolved, and single molecule FRET under physiological conditions. These studies indicate that the modified MBs act similarly to the 2'OH MB. The investigated MBs bind with similar affinity to the RNA target. The 2'OMe MB binds with 3-fold better affinity than the 2'OH MB, while the 2'F binds with similar affinity and 2'H MBs binds with 3-fold less. Single molecule studies show a lack of MB dynamics observed in the minute timescale, as well as a large dynamic range in that the molecular beacon alone was primarily in the closed conformation and, in the presence of the target, the beacon was primarily in the open conformation. Effect of an Intermittent Administration of MnCl2 on the Dopaminergic System, Rabab A. Aoun, Madiha Khalid, Tiffany A. Mathews, Wayne State University, Detroit, MI Overexposure to manganese (Mn), an essential trace element, results in a neurological disorder known as manganism, which resembles Parkinson's disease. Since the similarities between the two disorders are most likely due to their site of action in dopamine-rich areas of the basal ganglia, research has largely focused on the biochemical and behavioral alterations associated Mn toxicity on dopamine (DA). Following an established protocol by Dodd et al., (Int. J. Toxicol., 2005, 24: 389-97) C57/Bl6 mice (20-25 g) were administered with MnCl2 (50 mg/kg, s.c.) on days 1, 4, and 7. Brain, liver, and kidney homogenates were evaluated for Mn accumulation using graphite atomic absorption spectrometry. Additionally, tissue content levels of monoamines (DA, NE, 5-HT), their metabolites (DOPAC, 3-MT, HVA, and 5-HIAA), and amino acids (GABA and Glu) were evaluated in the caudate-putamen (CPu), midbrain, frontal cortex, and hippocampus using HPLC coupled to electrochemical or fluorescence detection. Significant accumulation of Mn was seen 24 h (day 8) following the last Mn injection in all brain regions, but no difference in neurotransmitter levels was observed. However, 3 weeks (day 28) following the last Mn injection, a significant decrease in DA and its metabolite DOPAC was observed in the frontal cortex of Mn-treated mice. Surprisingly, no differences were observed in DA or its metabolite DOPAC in the CPu on day 28. These findings suggest that DAergic signaling in the frontal cortex may be more susceptible to Mn toxicity compared to other DA-rich regions, such as the CPu. However, in vitro fast-scan cyclic voltammetry preliminary results revealed an initial decrease in DA release, which recovered to control levels by day 28, suggesting Mn does acutely alter DA neurotransmission in the CPu. Future studies will further characterize the time course effect of Mn on DA.
Brain-derived Neurotrophic Factor Deficient Mice Appear to be
Hyperdopaminergic, In addition to acting as a nerve growth factor, brain-derived neurotrophic factor (BDNF) has been proposed to modulate synaptic neurotransmission. For example, acute administration of exogenous BDNF in adult rodents was shown to influence striatal dopamine release and modulate dopamine-related behaviors including locomotor stimulation. In the present study, we evaluated if low endogenous levels of BDNF alter dopamine system function using in vivo microdialysis and in vitro fast scan cyclic voltammetry in the dorsal striatum of heterozygote BDNF mice (BDNF+/-). Using the zero net flux method, apparent extracellular dopamine levels were found to be elevated in the BDNF+/- mice compared to wildtype controls (12 vs. 5 nM, p < 0.001). Furthermore, dopamine extraction fraction values, which estimate neurotransmitter clearance from the extracellular space, were significantly different in these two groups of mice (p < 0.05). Using in vitro voltammetry, it appears that extracellular dopamine levels are increased due to a decrease in dopamine reuptake rates in the BDNF+/- mice. Additionally, electrically-evoked dopamine appears to be decreased in BDNF+/- mice. Finally, BDNF+/- mice are being evaluated after an acute administration of known drugs of abuse such as methamphetamine and ethanol. Preliminary ethanol studies show that the BDNF+/- mice have a blunted response to a systemic injection of ethanol. Using both microdialysis and voltammetry reveals that BDNF-deficient mice are hyperdopaminergic. Future work will use both of these techniques to further probe potential subtleties within the dopaminergic system. Ion/Ion Reactions in Analytical Mass Spectrometry: Beyond Single Proton or Single Electron Transfer, Plenary Lecture by the 2008 ANACHEM Award Recipient: Scott A. McLuckey, Kerry H. Hassell, Marija Mentinova and Hongling Han,Department of Chemistry,Purdue University,West Lafayette, IN 47907-2084 Gas-phase ion/ion reactions involving multiply-charged ions is a relatively new class of chemical reactions that can be used in conjunction with mass spectrometry to address issues in (bio)chemical analysis. Many examples of the utility of ion/ion reactions have already been demonstrated and several instrument vendors now offer products that make ion/ion reactions accessible to the analyst. These examples are dominated by reactions that involve single small charged particle transfers, i.e., either proton transfer or electron transfer. Proton transfer reactions are particularly useful for charge state manipulation whereas electron transfer reactions are useful as a dissociation method (i.e., electron transfer dissociation). The range of reactions that can occur in ion/ion chemistry, however, extends far beyond single electron or single proton transfer. This presentation emphasizes reactions other than single proton or single electron transfer. These include, for example, metal ion transfer, reactions that involve the inversion of charge of the analyte ion, and selective reactions that result in covalent bond formation. Applications of these ion types are far less mature than those of the single electron or proton transfer. However, they offer new opportunities/possibilities for enhancing specificity and sensitivity in analytical mass spectrometry.
Recent Advances in Academic MS Research Structures and Binding Energies of Noncovalent Complexes of Peptidomimetic Protonated Nitrogen Bases with 18-Crown-6, Yu Chen, M.T. Rogers, Wayne State University, Detroit, MI Protein structure is one of the keys to understanding function or malfunction in biological systems at the molecular level. Methods using agents that selectively bind to peptides and proteins through complexation enable rapid identification and characterization of protein sequence, structure, and function. For example, selective noncovalent adduct protein probing (SNAPP) utilizes noncovalent recognition of basic amino acid residues, and in particular lysine residues, by 18C6 to monitor changes in protein structure. In this study, absolute 18C6 affinities of six model protonated bases that serve as mimics for the side-chain of lysine, histidine arginine and n-terminus of peptides are determined by guided ion beam tandem mass spectrometry. The model bases examined in the present study include: imidazole (IMID), 4-methylimidazole (4MeIMID), n-butylamine (NBA), 1,5-diamino pentane (DAP), isopropylamine (IPA) and methylguanidine (MGD). The kinetic-energy-dependent cross sections for collision-induced dissociation (CID) of six (18C6)H+(B) complexes are analyzed using an empirical threshold law to extract absolute 0 and 298 K bond dissociation energies (BDEs) after accounting for the effects of multiple collisions, kinetic and internal energy distributions of the reactants, and unimolecular dissociation lifetimes. Theoretical electronic structure calculations are performed to determine stable geometries and energetics for neutral and protonated 18C6 and the model bases as well as the proton bound (18C6)H+(B) complexes. Atmospheric Pressure Femtosecond Laser Imaging Mass Spectrometry,Yves Coello, A. Daniel Jones, Tissa Gunaratne and Marcos Dantus, Michigan State University, East Lansing, Michigan We present a novel imaging mass spectrometry (IMS) approach that uses near-infrared femtosecond laser pulses for direct desorption and ionization of sample constituents under ambient conditions. The method offers significant advantages over matrix-assisted laser desorption/ionization (MALDI) imaging by eliminating the need for a laser-absorbing matrix, being suitable for atmospheric pressure (AP) sampling, and by providing 10 µm spatial resolution, as we demonstrate with a chemical image of vegetable cells. Mass spectrometry imaging (MSI) is becoming an increasingly important tool in many areas of the life sciences because of its ability to image specific analytes in biological samples [1]. While most experiments have been traditionally performed at vacuum conditions by either secondary ion mass spectrometry (SIMS) or ultraviolet (UV) MALDI [2,3], there is currently an increasing interest in AP MSI techniques because they allow the study of live tissues. AP IMS techniques include desorption electrospray ionization (DESI), laser ablation electrospray ionization (LAESI), and infrared (IR) MALDI [4-6], which provide lateral resolutions between 200 and 400 µm. Unfortunately, these resolutions are still insufficient to analyze the distribution of molecules in cellular and sub cellular structures. The main motivation of this work is to provide a high resolution AP IMS approach able to analyze biological samples at the cellular level. Furthermore, given that femtosecond laser pulses can ablate and ionize sample constituents by direct nonlinear laser-molecule interaction, the need for a laser absorbing matrix is eliminated, making sample preparation and handling significantly simpler. We expect to improve our current 10 µm spatial resolution by synchronizing the laser pulses with the pusher pulses in the mass spectrometer. This will allow higher ion detection efficiency and thus higher lateral resolution. Fig. 1. Chemical image of onion skin cells. The image shows the distribution of glucose in the sample (m/z 179, corresponding to [M-H]- of glucose in negative ion mode). The inset shows the optical image. The lateral resolution is 10 µm. (Not Shown)
[1] McDonnell, L. A.; Heeren, R. M. A., Imaging mass spectrometry. Mass
Spectrometry Reviews 2007, 26, (4), 606-643.
Probing the Effects of Protonation on the Structure and Stability of Uracil,
Thiouracils, and Methylthiouracils via IRMPD Action Ion Spectroscopy and
Theoretical Calculation, Yuan-wei Nei1 , Tolulope E. Akinyemi1, Jeffrey
D. Steill2 , Jos Oomens2 , and M. T. Rodgers1*, Uracil and thiouracils are of great interest due to their biological roles, pharmacological applications, and spectroscopic activities. Our laboratory had extensively studied these systems using guided ion beam tandem mass spectrometry (GIBMS) techniques and electronic structure calculations to measure their bond dissociation energies and model their geometric behavior in the gas phase. Recently, infrared multiphoton dissociation (IRMPD) action spectra of protonated and metal complexed uracil, thioketo substituted uracils (2SU, 4SU, and 2,4-dSU), and methyl thioketo substituted uracils (5Me2SU and 6Me2SU) were measured at the FOM Institute for Plasma Physics, "Rijnhuizen" in the Netherlands in an attempt to better characterize the structures and stability of these species. The protonated species will be the main focus of this presentation. Protonated uracil, thiouracils, and methylated thiouracils were introduced into the gas phase using an electrospray ionization source (ESI). These species were then injected into an ion cyclotron resonance (ICR) magnetic trap for mass analyses. Ion of interest was isolated using stored waveform inverse Fourier transform (SWIFT) techniques. Low energy activated dissociation pathways were then probed by IRMPD action spectroscopy using a free electron laser (FEL) as the radiation source. Utilizing the wide wavelength tunability of the FEL, IRMPD action spectra were collected from the relative intensities of low energy dissociation products of these species as a function of FEL wavelength over the IR fingerprint region extending from 1050 to 1850 cm-1. The experimental spectra were then compared to theoretical linear IR spectra obtained from density functional theory (DFT) calculations using the B3LYP/6-311+G(2d,2p)//B3LYP/6-31G* level of theory for structures, frequencies, and energetics of these systems. Agreement between the experimental IRMPD spectra and the calculated linear spectra for the ground state structures were excellent for several systems, while in other cases somewhat surprising results were found. A Sulfonium Ion Containing Protein Modification Reagent and Tandem Mass Spectrometry for the Study of Protein Surface Accessibility and Protein Interactions, Xiao Zhou1 and Gavin E. Reid1,2, 1Department of Chemistry and 2Department of Biochemistry and Molecular Biology. Michigan State University, East Lansing, MI 48824 Protein surfaces have great importance for the study of protein folding, protein-ligand binding and protein-protein interactions. Amino-acid specific covalent labeling coupled with mass spectrometry (MS) analysis has proven to be a useful tool for protein surface mapping. The reactivity of amino acids towards the labeling reagent depends not only on the intrinsic chemical property but also the solvent accessibility of the amino acids. Analysis of the modified peptides by MS and tandem mass spectrometry (MS/MS) can reveal the extent of modification, the modification sites, and the sequence of the peptides of interests. However, a common problem associated with current MS/MS based protein labeling strategies is that when a complicated peptide mixture resulting from a large protein complex is analyzed, the modified peptides may be difficult to detect amongst the largely unmodified peptide population. Here, an amine-specific "fixed charge" sulfonium ion containing protein modification reagent, S, S'-dimethyl thiobutanolhydroxysuccinimide ester sulfonium iodide, was developed. This protein modification reagent gives rise to an exclusive neutral loss upon collision induced dissociation (CID), thereby enabling the simplified identification of modified peptides from within complex mixtures. Localization of the modification sites and peptide sequence information can be obtained by further fragmentation of the neutral loss product ion. The initial success of this sulfonium ion containing reagent has been demonstrated on a model protein, CRABP II.
ESI-MS Detection of Degmented Flow and Application to Fraction Collection
from Capillary Liquid Chromatography, Qiang Li1, Jian Pei1, Mike S. Lee3,
Gary A. Valaskovic4 and Robert T. Kennedy1,2, Droplets or plugs within multiphase microfluidic systems have rapidly gained interest as a way to manipulate samples and chemical reactions on the femtoliter to microliter scale. Chemical analysis of the plugs remains a challenge. We have discovered that nanoliter plugs of sample separated by air or oil can be analyzed by electrospray ionization mass spectrometry (ESI-MS) when pumped directly into a fused silica nanospray emitter tip. Using leucine-enkephalin in methanol and 1% acetic acid in water (50:50 v:v) as a model sample, we found carry-over between plugs was < 0.1% and relative standard deviation of signal for a series of plugs was 3%. With optimal ESI voltage at 1.5 kV and flow rate range from 20 to 2000 nL/min, aqueous plugs underwent ESI while oil plugs were dripped off the emitter tip or extracted out with side hydrophobic Teflon tubing. Droplet sample analysis rates of 2.3 Hz were achieved by pumping 8 nL samples separated by 5 nL oil gaps in a 150 µm inner diameter (i.d.) tube. An application of this technology is for fraction collection from capillary LC and off-line ESI-MS analysis. Using a 100 µm i.d. T junction, effluents from a capillary LC separation were segmented by oil. With this approach, chemically separated components were preserved as nanoliter level plugs and could be stored and transported easily. The segmented flows were than analyzed off-line on a linear ion trap MS, leading to results as consistent as traditional on-line analysis. Peak parking of selected peaks could be done by changing the infusion rate of the segmented flow to obtain more structural information of the samples. The system provides a robust, rapid, and information-rich method for chemical analysis of sample in segmented flow systems. Spray Desorption Collection, Afrand Kamali, Andre Venter, Chemistry Department,Western Michigan University, Kalamazoo, 49008 We are reporting on the successful development of a new surface sampling technique called Spray Desorption Collection (SDC). Spray Desorption collection (SDC), is based on desorption electro-spray ionization (DESI) but unlike DESI where desorbed analytes are analyzed immediately, SDC collects desorbed analytes onto a suitable surface for subsequent analysis. Surface sampling for mass spectrometric analysis is typically achieved by swab-wiping or liquid-washing. These approaches tend to distribute sample material over large areas or volumes. Pre-concentration by sampling large areas for dispersed, low concentration surface analytes are difficult to impossible. During desorption electrospray ionization (DESI), surface material is desorped by solvent spray from surfaces and sampled into a mass spectrometer (MS). By replacing the mass spectrometer with a suitable collection surface, the analyte containing, spray is captured. Repeated collection on the same collection surface, from multiple sample surfaces, leads to pronounced pre-concentration. After sampling, the collection surface can be analyzed by ambient (direct) desorption MS, or the collected material can be recovered into any suitable solvent for analysis by other methods. In this presentation we discuss collection efficiencies and the optimization of various parameters . We also demonstrate applications of SDC to collect samples for subsequent analysis by DESI-MS , GC-MS and UV-Vis spectrometry. POSTER SESSION Spray Desorption Collection (SDC) - A Novel Sampling Technique Coupled with UV Absorbance Spectrometer for Nano Particle Analysis, Shashank Jain, Andre Venter, Western Michigan University, Kalamazoo, 49008 Over the last few decades there has been a rush for the synthesis of various types of nanoparticles ranging from 1nm to 100nm.Owing to their amazing physicochemical properties nanoparticles find application in various fields. With the increasing number of applications of nanomaterials, the risk of exposure to humans and to the environment is increasing. As a result measurement of occupational, consumer and environmental exposure to nanomaterial's and mitigation is of great interest. In order to reduce uncertainty about risks posed by nanomaterials, the development of new methods to detect nanomaterials in biological matrices, the environment and the workplace, has been identified as key to addressing some existing problems. There is an urgent need of fast, efficient and reliable analytical techniques for the analysis and separation of nanoparticles from the environment. The natural state for nanomaterials is in a dry powder form, presenting particular sample-collection challenges. We report on the use of a novel sampling technique for the collection and subsequent analysis of nanoparticles from surfaces. Spray Desorption Collection (SDC) is based on desorption electrospray ionization and functions by directing a pneumatically assisted solvent spray at a sample surface. Surface materials, soluble in the spray solvent, are carried away when fast moving droplets collide with and ricochet off the surface. Soluble surface material and solvent is collected on a surface where the solvent evaporates while the analyte is preconcentrated. After collection, the analyte (nanoparticles in this case) can be analyzed by any suitable analytical technique. Here we demonstrate the use of UV Absorbance for analysis. Cation Aza-crown Complexes: Determination of Structures and Bond Dissociation Energies Using Guided Ion Beam Tandem Mass Spectrometry and Quantum Chemical Calculations.+Calvin Austin and M.T. Rodgers, Department of Chemistry, Wayne State University, Detroit, MI, 48202 Macrocyclic complexes have been useful in understanding many systems encountered in biology along with having widespread use in analytical, pharmaceutical, and synthetic chemistry. In the present study our goal is to provide a quantitative experimental and theoretical description of cation-aza-crown interactions. Quantum chemical calculations are employed to characterize the structures and stabilities of the isolated cations and aza-crowns as well as noncovalently bound complexes comprised of these species. Guided ion beam tandem mass spectrometry techniques are used to characterize the energy dependence of the collision-induced dissociation of these cation-aza-crown complexes. Quantitative thermochemical analysis of experimental data then provides accurate binding energies of the cation-aza-crown complexes. We plan to investigate a variety of cations including: alkali metal cations, transition metal cations, and organic cations. Likewise we will investigate a variety of aza-crowns that vary in size, number of donor atoms, linkage geometries, and substitution. Initial studies examine Li+, Na+, K+, Rb+, and Cs+ interacting with multiple dimethylamine (DMA) ligands or a single 1,4,7,10-tetraazacyclododecane ([12]aneN4), 1,4,7,10,13-pentaazacyclopentadecane ([15]aneN5), or 1,4,7,10,13,16-hexaazacyclooctadecane ([18]aneN6). The ground state structures of the alkali metal cation complexed with one to four DMA ligand has been investigated. By dissecting [12]aneN4, we are able to look at the interactions of the cation-ligand complex in a simpler environment to better evaluate the contributions from: the number of metal cation donor interactions, bonding in the complexes, and steric effects. This approach allows us to examine the nonconvalent interactions of a simple ion-molecule complex in detail where the selectivity and additive properties are identified and applied to the larger ring systems. Accumulation of Manganese in Kidney, Liver, and Brain Upon Intermittent Subcutaneous Injections of MnCl2, Rabab A. Aoun, Madiha Khalid, Tiffany A. Mathews, Wayne State University, Detroit MI 48202 Manganese (Mn), required for normal body functioning in trace amounts, has the potential to become neurologically damaging at higher concentrations. Similar to Parkinson's, the symptoms of the Mn overexposure disorder, manganism, appear to be primarily linked with motor activity and thus the dopamine (DA) system has been suggested to play a role in its onset. Our project focused on studying the effect of intermittent subcutaneous injections of MnCl2 on the kidneys, liver, and brain. C57/Bl6 mice were administered with MnCl2 (50 mg/kg, s.c.) on days 1, 4, and 7. Twenty four hours later, graphite atomic absorption was used to evaluate Mn accumulation in kidney, liver, and various brain region homogenates. Kidney and liver samples showed Mn accumulation; however, only certain dopamine-rich regions of the brain showed elevated Mn levels. HPLC was used to further evaluate the caudate-putamen (CPu), midbrain, and cortex for certain monoamines and their metabolites via tissue content studies. Only a decrease in DA and DOPAC was observed in the cortex 21 days after the final injection. Finally, fast scan cyclic voltammetry was used to test the effect of Mn on stimulated DA release and uptake in the CPu. No difference was observed between uptake rates; however, DA release varied as a function of time. Also, Quinpirole, a D2 receptor agonist, was used to evaluate the effect of Mn on the D2 receptor on day 8 and 28. Preliminary data shows a shift in EC50 values between each of these time points and their respective controls. Determining the Efficacy of Non-Destructive Pre-Treatment Methods on Known Age Textile Samples by Gas Chromatography-Mass Spectrometry Analysis, Deidre Hardemon and Ruth Ann Armitage, Eastern Michigan University, Ypsilanti, MI 48197 The purpose of this study is to evaluate the efficacy of a new pre-treatment method for removing contamination prior to nondestructive radiocarbon dating of textile samples. Soil contamination, in the form of humic acids, is generally present on or in any archaeological materials when they are removed from their burial contexts. It is important for the samples to be clean and free of humic acids prior to radiocarbon dating, as these contaminants will contribute to the measured radiocarbon age. Modern-age wool and linen samples were contaminated with 11,000-year-old humic acid by soaking them in solution for one month to simulate an underwater archaeological environment. We evaluated three pre-treatment methods: the standard - and destructive - acid-base-acid method; a pH 8 phosphate buffer; and water as a control. The solutions resulting from the cleaning steps were extracted, derivatized, and subjected to gas chromatography-mass spectrometry in order to quantify compounds like nonanedioic acid that are markers for humic acid contamination in these contexts. Plasma-chemical oxidation and radiocarbon analysis by accelerator mass spectrometry will be carried out on the cleaned textiles to determine the efficacy of the treatments: the difference between the measured age and known age can be used to determine the proportion of contamination in the sample. This will be compared to the GC-MS analysis to see how the two are correlated. The results of this work are an important step in applying this new dating technique to waterlogged and otherwise heavily contaminated archaeological textiles. Alterations in Dopamine Neurotransmitter Dynamics in Brain-derived Neurotrophic Factor Deficient Mice, K. E. Bosse, M. M. France, J. J. P. Roberts and T. A. Mathews, Wayne State University, Detroit, MI In addition to acting as a nerve growth factor, brain-derived neurotrophic factor (BDNF) has been proposed to modulate synaptic neurotransmission. For example, acute administration of exogenous BDNF in adult rodents was shown to influence striatal dopamine release and modulate dopamine-related behaviors including locomotor stimulation. In the present study, we evaluated if low endogenous levels of BDNF alter dopamine system function using in vivo microdialysis in the dorsal striatum of heterozygote BDNF mice (BDNF+/-). Using the zero net flux method, apparent extracellular dopamine levels were found to be elevated in the BDNF+/- mice compared to wildtype controls (11.89 ± 0.32 nM vs. 4.89 ± 0.39 nM, p < 0.001). Furthermore, dopamine extraction fraction values, which estimate neurotransmitter clearance from the extracellular space, were significantly different in these two groups of mice (p < 0.05). In BDNF+/- mice, perfusion of high potassium (120 mM) stimulated an approximate 10-fold increase in dopamine release compared to a 6-fold increase in wildtype controls. In contrast, no differences were observed in the striatal levels of L-DOPA tissue content or extracellular dopamine metabolites (DOPAC and HVA). These results indicate that in intact mouse striatum, BDNF tonically regulates dopaminergic activity through a mechanism which does not involve changes in dopamine synthesis or metabolism. Rather these data suggest BDNF+/- mice may have altered dopamine transporter-mediated function in the striatum. Allaria petiolata (Garlic mustard) Seed Characterization, Benedict Diatta, Shanmugam Puvanendran, Andre Venter, Western Michigan University, Kalamazoo, 49008
Allaria petiolata, also known as garlic mustard, a herb native to Europe,
was introduced to the North American continent as a culinary herb in the
1860s. Since the insects and fungi that feed on it in its native habitat
are not present in North America, it is out-competing many native plants.
It has since 2006 been listed as a noxious or restricted plant in many states
of the USA due to the devastating effect it has on native plant and insect
species [1]. Our aim is to find or develop a value added product that can
be produced from garlic mustard to help fund eradication efforts of this
invasive weed. Our first step is to determine the chemical composition of
Allaria petiolata at various stages of the plants' life cycle. In this poster
we present the complete analysis of garlic mustard seeds. Dried seed were
continually and sequentially extracted by petroleum ether, chloroform, methanol
and water using a soxhlet apparatus. The petroleum ether and chloroform extract
were analyzed by Gas Chromatography-Mass spectrometry. GC/MS was previously
used to determine the chemical composition of the volatile components of
Allaria Petiolata in plant material consisted of stems, flowers and leaves
[2] and we successfully replicated those results for seeds and found many
of the same components previously identified in the steams, leaves and flowers.
In addition, we also studied the acidic and basic water and alcohol soluble
compounds of garlic mustard by electrospray ionization (ESI) and desorption
electrospray ionization (DESI) mass spectrometry. The combined results of
all extraction fractions reveal a very interesting chemistry and many natural
compounds. Analysis of a Series of Late Medieval Korean Coins via Energy Dispersive X-Ray Fluorescence, Danielle Grashott, Elizabeth MacDonald, Stephanie Spohn, and Mark A. Benvenuto, University of Detroit Mercy, Detroit, MI 48221 A series of early nineteenth century Korean coins were analyzed using energy dispersive X-ray fluorescence (EDXRF) spectroscopy. The general morphology of each item was a round coin with a central, generally square hole, four characters on the obverse or head side, and two characters on the reverse or tails side. The total sample set was examined via EDXRF both on the obverses and reverses. The composition of the coins varied, but routinely included copper, lead and zinc as major components. Considerably smaller but significant amounts of antimony, tin, iron, and silver were present in the majority of the samples. The full findings are presented and discussed. In Vitro Evaluation of Dopamine D3 Receptors in Nucleus Accumbens Using Fast Scan Cyclic Voltammetry, Francis K. Maina, Katherine L. Logan, and Tiffany A. Mathews, Wayne State University, Detroit, MI, 48202 Dopamine (DA) has attracted much attention due to its involvement in: addiction, reward, movement, emotion, motivation, cognition, learning, Schizophrenia, Parkinson's disease, and attention deficit hyperactivity disorder (ADHD). The actions of DA are mediated by five receptor subtypes that can be grouped into two subfamilies, D1-like and D2-like. The D2-like family is known to control DA neurotransmission and is composed of D2, D3 and D4 receptors. The D3 receptors have a fairly restricted pattern of expression in the mammalian brain compared to other D2-like receptors. Specifically the localization of DA D3 receptors in the nucleus accumbens (NAc) core and shell is of particular interest because this brain region is often associated with the rewarding properties of drugs of abuse. Fast scan cyclic voltammetry (FSCV) was used to detect electrically stimulated DA release and uptake rate in 400 µm-thick coronal mouse brain slices containing the CPu and NAc. Increasing concentrations of quinpirole (D2 agonist) or (+)-PD 128907 (D3 agonist) were added to slices at 30-minute intervals. Quinpirole showed no difference in DA inhibition in the CPu and NAc, suggesting that D2 autoreceptor functionality is similar in the regions investigated. However, (+)-PD 128907 showed greater DA inhibition in the shell compared to CPu based on the EC50 values while no effect in uptake rate was observed. This preliminary study confirms that there are higher DA D3 receptor levels in the NAc versus the CPu. To confirm the selectivity of the agonists, sulpiride (D2 antagonist) and nafadotride (D3 antagonist) were used to reverse the agonist's effect. Methodological Study of Two Derivatization Methods for Proteinaceous Binders in Rock Paintings, Geneve Maxwell and Ruth Ann Armitage, Eastern Michigan University, Ypsilanti, MI 48197 In this study, two different derivatizing methods were used to characterize the proteinaceous materials in paint binders for both modern and archaeological samples. Each method involved solid phase extraction clean up, hydrolysis, and derivatization. Proteins were extracted using ammonia and were separated from the inorganic salts by using pipet tips loaded with C18 stationary phase. While the SPE step should remove inorganic salts that could interfere with hydrolysis, it also resulted in significant loss of protein, so much so that this technique was not applied to the modern or archaeological samples. Instead, the inorganic salts were removed and the proteins extracted using only the ammonia. The ammoniacal extract of the modern and authentic paints were hydrolyzed to produce amino acids. Two aliquots of hydrolysate were removed for derivatization. N-(O,S)-ethoxycarbonyl ethoxy esters of the amino acids were formed by reaction of one aliquot with ethyl chloroformate. The other aliquot was silylated with N-methyl-N-t-butyldimethylsilyl- trifluoroacetamide (MTBSTFA). The two derivatization methods were compared to determine which would yield more useful information, both qualitatively and quantitatively, about the proteinaceous binders when subjected to GC-MS. Ultimately, this work will aid archaeologists who study rock paintings in understanding both the age and composition of these unique artifacts. Qualitative and Quantitative Analyses of Lipidic Binders in Rock Paintings, Christina Phillips and Ruth Ann Armitage, Eastern Michigan University, Ypsilanti, MI 48197 In order to reliably determine the age of rock paintings with radiocarbon analysis, it is important to know as much as possible about the organic composition of the paint. One of the limitations in characterizing the binders in rock paintings by GC-MS is the amount of sample available for analysis. Different classes of compounds - lipids, proteins, carbohydrates - are found together in the natural substances used as binders. Some methods, like thermally assisted hydrolysis/methylation-GC-MS, yield qualitative information about al three classes in a single analysis. Quantitative analysis requires a more targeted approach. This study focuses on using the residue left after extracting the proteins from modern and archaeological rock painting binders for GC-MS analysis of the fatty acids. Authentic rock art paints and modern binders prepared in the laboratory were treated with ammonia to extract proteins. The remaining residue was saponified in ethanolic KOH (10%) and the resulting free fatty acids were extracted into diethyl ether. An aliquot of the ether extract was then derivatized to form trimethylsilyl esters of the extracted fatty acids, which were then subjected to GC-MS for quantitative analysis. The identities and quantities of the fatty acids present in the modern binders will help us to better understand the nature of the archaeological materials. Gas-Phase Conformations of Cationized Mononucleotides Determined by Infrared Multiphoton Dissociation Spectroscopy and Theoretical Modeling, Ranran Wu and M. T. Rodgers*, Wayne State University, Detroit, MI, 48202 Metal cations often exert a significant influence on nucleic acid structures and genetic information transfer. By interacting with the phosphate group, nucleobases and the sugar ring, metal cations may stabilize the structures of DNA. Metal cation-nucleic acid interactions are of great interest because metal cations and metal cation-ligand complexes can bind to DNA to regulate gene expression and thus can be used as tools for molecular biology studies. The gas-phase conformations of cationized (protonated and metal-cationized) mononucleotides are examined using infrared multiple photon dissociation (IRMPD) spectroscopy utilizing light generated by a free electron laser and electronic structure theory calculations. Measured IRMPD action spectra of the cationized complexes are compared to the theoretical linear IR spectra calculated at the B3LYP/6-31G (d) level of theory in order to identify the structures present in the experimental observation. Effects of different metal cations on the conformations and the stabilities of the cationized mononucleotides are examined. Investigation of the metal cation interactions within the mononucleotides are performed to better understand the importance of cationization in biological processes, in which metal cation-nucleic acid interactions play roles. Pharmaceutical Cleaning Validation by Spray Desorption Collection, Kevin Douglas, Shashank Jain, Andre Venter, Western Michigan University, Kalamazoo, 49008 Surface Desorption Collection (SDC) is a recently developed surface sampling technique that uses desorption electrospray ionization (DESI) offline to transfer analyte from a surface to a portable substrate. Subsequent analysis is then dependent on the substrate; DESI-MS, ion mobility spectrometry (IMS), and UV/Vis have each been used to successfully analyze samples collected by SDC. Compared to other surface sampling techniques such as swabbing and solvent rinsing, SDC preconcentrates the analyte while eliminating the need for sample preparation. Since any size surface area can be scanned and collected on a single substrate, SDC is capable of analyzing surfaces with extremely low analyte surface concentration. For this experiment, SDC was tested as a viable surface sampling technique for use in pharmaceutical cleaning validation. Phenylephrine HCl in pharmaceutical matrix was loaded onto a stainless steel surface and collected by SDC on PTFE substrates for analysis by IMS. The results were then compared with those from samples acquired by traditional swab techniques followed by solvent extraction before IMS analysis. Fast Analysis of GABA and Glutamate in Dialysis Samples Using a Monolithic Column, Stella Wisidagama and Tiffany A. Mathews, Wayne State University, Detroit, MI, 48202 Using a monolithic column and gradient high performance liquid chromatography (HPLC) method was developed for a rapid and simultaneous detection of glutamate and gamma-aminobutyric acid (GABA). Glutamate and GABA levels were measured in the caudate-putamen using the in vivo technique - microdialysis. The dialysate samples were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) and analyzed by gradient HPLC (monolithic column, C18, 100 x 4.5 mm) with fluorescence detection. GABA and glutamate were eluted using gradient conditions, and they were separated with a run time of less than 5 minutes. The average extracellular concentrations of GABA and glutamate in the caudate-putamen were approximately 0.06 and 0.44 µM, respectively. Current work is focusing on evaluating the effect of reduced levels of brain derived neurotrophic factor (BDNF) on extracellular glutamate and GABA levels in the caudate-putamen. Preliminary studies, suggest that there is no difference between glutamate and GABA levels in mice with reduced levels of BDNF and their wildtype littermates. Finally, to demonstrate the feasibility of this method, extracellular GABA and glutamate changes in the caudate-putamen were monitored following stimulation with high potassium artificial cerebral spinal fluid.
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