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  • The Limp-2/scarb2 Binding Motif On Acid Beta-glucosidase Basic And Applied Implications For Gaucher Disease And Associated Neurodegenerative Diseases

    Type:
    Article
    Description/Abstract:
    Background: Acid -glucosidase is trafficked to the lysosome by LIMP-2. Results: A unique 11-amino acid sequence on acid -glucosidase was critical for its LIMP-2-dependent targeting to the lysosome. Conclusion: This sequence is essential for oligosaccharide-independent targeting of synthesized acid -glucosidase to the lysosome. Significance: Modification of this sequence has basic/therapeutic implications for Gaucher disease and its comorbidities (e.g. Parkinson disease).
    Creator/Author:
    Grabowski, Gregory A.; Haffey, Wendy D.; Greis, Kenneth D., and Llou, Benjamin
    Submitter:
    Kenneth Greis
    Date Uploaded:
    03/03/2017
    Date Modified:
    04/07/2017
    Date Created:
    2014-09
    License:
    All rights reserved

  • Initial Validation of a Novel Protein Biomarker Panel for Active Pediatric Lupus Nephritis

    Type:
    Article
    Description/Abstract:
    Lupus nephritis (LN) is among the main determinants of poor prognosis in systemic lupus erythematosus (SLE). The objective of this study was to 1) isolate and identify proteins contained in the LN urinary protein signature (PS) of children with SLE; 2) assess the usefulness of the PS-proteins for detecting activity of LN over time. Using surface-enhanced or matrix assisted laser desorption/ ionization time of flight mass spectrometry, the proteins contained in the LN urinary PS were identified. They were transferrin (Tf), ceruloplasmin (Cp), α1-acid-glycoprotein (AGP), lipocalintype prostaglandin-D synthetase (L-PGDS), albumin and albumin-related fragments. Serial plasma and urine samples were analyzed using immunonephelometry or ELISA in 98 children with SLE (78% African-American) and 30 controls with juvenile idiopathic arthritis. All urinary PS-proteins were significantly higher with active versus inactive LN or in patients without LN (all p<0.005), and their combined area under the receiver operating characteristic curve was 0.85. As early as 3 months before a clinical diagnosis of worsening LN, significant increases of urinary Tf, AGP (both p < 0.0001) and L-PGDS (p < 0.01) occurred, indicating that these PS-proteins are biomarkers of LN activity and may help anticipate the future course of LN. Systemic Lupus Erythematosus (SLE) is an inflammatory autoimmune disease and lupus nephritis (LN) is one of the main determinants of poor prognosis (1). Currently, LN is gauged by measuring circulating and excreted indicators of renal dysfunction, with supporting information from kidney biopsies. The latter constitute the current standard for diagnosing LN, providing a direct assessment of the presence, severity and activity of LN, and the degree of renal damage (2). Due to the invasive nature of kidney biopsies, clinicians base LN activity and its therapy on the results of urinary protein excretion, urinary sediment, creatinine clearance and serum albumin. These traditional markers are not accurate in assessing whether active LN is present or not, and none of them is predictive, i.e. can anticipate the course of LN. Using Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF MS) technology, we previously identified a LN urinary protein signature (PS), consisting of eight candidate biomarkers at the mass-to-charge ratios (m/z) of 2.763, 22, 23, 44, 56, 79, 100, and 133 kDa (3). In this study, we present the identification of the specific proteins contained in this PS of children with LN. We further assayed plasma and urine samples of SLE patients and controls with juvenile idiopathic arthritis (JIA) to investigate the concurrent and predictive validity of the PS-proteins to serve as biomarkers of LN activity.
    Creator/Author:
    Tucker, Lori; Brunner, Hermine I.; Wiers, Kristina; Olson, Judyann; Suzuki, Michiko; Onel, Karen; Ying, Jun; Greis, Kenneth D.; Devarajan, Prasad; Haines, Kathleen; O'Neil, Kathleen M.; Silverman, Earl D.; Klein-Gitelman, Marisa S., and Brooks, Elizabeth B.
    Submitter:
    Kenneth Greis
    Date Uploaded:
    03/03/2017
    Date Modified:
    04/07/2017
    Date Created:
    2009-02
    License:
    All rights reserved

  • Advanced hair damage model from ultra-violet radiation in the presence of copper

    Type:
    Article
    Description/Abstract:
    OBJECTIVE: Damage to hair from UV exposure has been well reported in the literature and is known to be a highly complex process involving initiation via absorption of UV light followed by formation and propagation of reactive oxygen species (ROS). The objective of this work was to understand these mechanisms, explain the role of copper in accelerating the formation of ROS and identify strategies to reduce the hair damage caused by these reactive species. METHODS: The location of copper in hair was measured by Transmission electron microscopy–(TEM) X-ray energy dispersive spectroscopy (XEDS) and levels measured by ICP-OES. Protein changes were measured as total protein loss via the Lowry assay, and MALDI ToF was used to identify the biomarker protein fragments. TBARS assay was used to measure lipid peroxide formation. Sensory methods and dry combing friction were used to measure hair damage due to copper and UV exposure and to demonstrate the efficacy of N,N’ ethylenediamine disuccinic acid (EDDS) and histidine chelants to reduce this damage. RESULTS: In this work, a biomarker protein fragment formed during UV exposure is identified using mass spectrometry. This fragment originates from the calcium-binding protein S100A3. Also shown is the accelerated formation of this peptide fragment in hair containing low levels of copper absorbed from hair during washing with tap water containing copper ions. Transmission electron microscopy (TEM) X-ray energy dispersive spectroscopy (XEDS) studies indicate copper is located in the sulphur-poor endo-cuticle region, a region where the S100A3 protein is concentrated. A mechanism for formation of this peptide fragment is proposed in addition to the possible role of lipids in UV oxidation. A shampoo and conditioner containing chelants (EDDS in shampoo and histidine in conditioner) is shown to reduce copper uptake from tap water and reduce protein loss and formation of S100A3 protein fragment. In addition, the long-term consequences of UV oxidation and additional damage induced by copper are illustrated in a fourmonth wear study where hair was treated with a consumer relevant protocol of hair colouring treatments, UV exposure and regular shampoo and conditioning. CONCLUSIONS: The role of copper in accelerating UV damage to hair has been demonstrated as well as the ability of chelants such as EDDS and histidine in shampoo and conditioner products to reduce this damage.
    Creator/Author:
    McComb, D. W.; Marsh, J. M.; Sun, Y.; Chaudhary, T.; Flagler, M. J.; Coderch, L.; Mamak, M.; Greis, Kenneth D.; Rubio, L.; Williams, R. E. A., and Davis, M. G.
    Submitter:
    Kenneth Greis
    Date Uploaded:
    03/03/2017
    Date Modified:
    04/07/2017
    Date Created:
    2015-04
    License:
    All rights reserved

  • Transformations in pharmaceutical research and development, driven by innovations in multidimensional mass spectrometry-based technologies

    Type:
    Article
    Description/Abstract:
    In the pharmaceutical industry, there is a tremendous need for qualitative and quantitative analysis of target analytes such as peptides, proteins, drugs, metabolites, biomarkers, impurities, and degradation products in various mixtures including synthetic reactions, in vitro cultures, biological fluids, drug substances, finished products, and many others. To provide adequate specificity for analysis in these complex mixtures, multidimensional analytical techniques are required. Mass spectrometry plays a central role in many of these multidimensional approaches to mixture analysis because it provides an unparalleled combination of sensitivity and specificity that is useful for both molecular identification and quantitative applications. Recent innovations in mass spectrometry and industrial implementation of these advances have transformed many aspects of pharmaceutical research and development. Data that were previously unattainable, or were not collected due to exorbitant cost or time constraints, can now be obtained using mass spectrometry-based technologies. The impact of these innovations has been most dramatically felt in early stages of discovery, as more data are available to make critical decisions, such as selecting compounds for advancement to costly preclinical and clinical trials. New MS technologies have also accelerated the progression of drug candidates through development and toward regulatory approval. Here, five major categories of pharmaceutical applications of mass spectrometry are reviewed. They are new chemical entity characterization, biomacromolecule characterization, bioanalytical quantitation, metabolite identification, and impurity and degradation product identification. A brief historical perspective and evolution of technologies for each application area are presented. Those discussions are followed with a description of the current strategies for implementation of the tremendous capabilities of the state-of-the-art approaches, along with representative applications. In addition, emerging technologies for each application area are presented to indicate the future directions of instrumentation for mixture analysis in the pharmaceutical industry. (Int J Mass Spectrom 212 (2001) 135–196) © 2001 Elsevier Science B.V.
    Creator/Author:
    Harbol, Kevin L.; Greis, Kenneth D.; Hoke, Steven H.; Morand, Kenneth L.; Baker, Timothy R., and Dobson, Roy L. M.
    Submitter:
    Kenneth Greis
    Date Uploaded:
    03/03/2017
    Date Modified:
    04/07/2017
    Date Created:
    2001-11
    License:
    All rights reserved

  • Heparin-Binding Motifs and Biofilm Formation by Candida albicans

    Type:
    Article
    Description/Abstract:
    Candida albicans is a leading pathogen in infections of central venous catheters, which are frequently infused with heparin. Binding of C. albicans to medically relevant concentrations of soluble and plate-bound heparin was demonstrable by confocal microscopy and enzyme-linked immunosorbent assay (ELISA). A sequencebased search identified 34 C. albicans surface proteins containing ≥1 match to linear heparin-binding motifs. The virulence factor Int1 contained the most putative heparin-binding motifs (n = 5); peptides encompassing 2 of 5 motifs bound to heparin-Sepharose. Alanine substitution of lysine residues K805/K806 in 804QKKHQIHK811 (motif 1 of Int1) markedly attenuated biofilm formation in central venous catheters in rats, whereas alanine substitution of K1595/R1596 in 1593FKKRFFKL1600 (motif 4 of Int1) did not impair biofilm formation. Affinity-purified immunoglobulin G (IgG) recognizing motif 1 abolished biofilm formation in central venous catheters; preimmune IgG had no effect. After heparin treatment of C. albicans, soluble peptides from multiple C. albicans surface proteins were detected, such as Eno1, Pgk1, Tdh3, and Ssa1/2 but not Int1, suggesting that heparin changes candidal surface structures and may modify some antigens critical for immune recognition. These studies define a new mechanism of biofilm formation for C. albicans and a novel strategy for inhibiting catheter-associated biofilms.
    Creator/Author:
    Andes, David R.; Lu, Jason Long; Tan, Queenie K., G.; Hostetter, Margaret K.; Greis, Kenneth D.; Zhang, Minlu; Orsborn, Kris I.; Porollo, Alexey, and Green, Julianne V.
    Submitter:
    Kenneth Greis
    Date Uploaded:
    03/03/2017
    Date Modified:
    04/07/2017
    Date Created:
    2013-07
    License:
    All rights reserved

  • Systematic evaluation of data-independent acquisition for sensitive and reproducible proteomics—a prototype design for a single injection assay

    Type:
    Article
    Description/Abstract:
    Data-independent acquisition (DIA)-based proteomics has become increasingly complicated in recent years because of the vast number of workflows described, coupled with a lack of studies indicating a rational framework for selecting effective settings to use. To address this issue and provide a resource for the proteomics community, we compared 12 DIA methods that assay tryptic peptides using various mass-isolation windows. Our findings indicate that the most sensitive single injection LC-DIA method uses 6 m/z isolation windows to analyze the densely populated tryptic peptide range from 450 to 730 m/z, which allowed quantification of 4465 Escherichia coli peptides. In contrast, using the sequential windowed acquisition of all theoretical fragmentions (SWATH) approach with 26 m/z isolation windows across the entire 400–1200 m/z range, allowed quantification of only 3309 peptides. This reduced sensitivity with 26 m/z windows is caused by an increase in co-eluting compounds with similar precursor values detected in the same tandemMS spectra, which lowers the signal-to-noise of peptide fragment-ion chromatograms and reduces the amount of low abundance peptides that can be quantified from 410 to 920 m/z. Above 920 m/z, more peptides were quantified with 26 m/z windows because of substantial peptide 13C isotope distributions that parse peptide ions into separate isolation windows. Because reproducible quantification has been a long-standing aimof quantitative proteomics, and is a socalled trait of DIA, we sought to determine whether precursor-level chromatograms used in some methods rather than their fragment-level counterparts have similar precision. Our data show that extracted fragment-ion chromatograms are the reason DIA provides superior reproducibility. Copyright © 2015 John Wiley & Sons, Ltd.
    Creator/Author:
    Haffey, Wendy D.; Norris, Jeremy L.; McCullumsmith, Robert E.; Heaven, Michael R.; Cobbs, Archie L.; Funk, Adam J., and Greis, Kenneth D.
    Submitter:
    Kenneth Greis
    Date Uploaded:
    03/03/2017
    Date Modified:
    04/07/2017
    Date Created:
    2016-01
    License:
    All rights reserved

  • Development of an Inhibitor Screening Platform via Mass Spectrometry

    Type:
    Article
    Description/Abstract:
    Commonly used methods for isolated enzyme inhibitor screening typically rely on fluorescent or chemiluminescent detection techniques that are often indirect and/or coupled assays. Mass spectrometry (MS) has been widely reported for measuring the conversion of substrates to products for enzyme assays and has more recently been demonstrated as an alternative readout system for inhibitor screening. In this report, a high-throughput mass spectrometry (HTMS) readout platform, based on the direct measurement of substrate conversion to product, is presented. The rapid ionization and desorption features of a new generation matrix-assisted laser desorption ionization-triple quadrupole (MALDI-QqQ) mass spectrometer are shown to improve the speed of analysis to greater than 1 sample per second while maintaining excellent Z′ values. Furthermore, the readout was validated by demonstrating the ability to measure IC50 values for several known kinase inhibitors against cyclic AMP–dependent protein kinase (PKA). Finally, when the assay performance was compared with a common ADPaccumulation readout system, this HTMS approach produced better signal-to-background ratios, higher Z′ values, and a reagent cost of about $0.03 per well compared with about $0.60 per well for the fluorescence assay. Collectively, these data demonstrate that a MALDI-QqQ-MS–based readout platform offers significant advantages over the commonly used assays in terms of speed, sensitivity, reproducibility, and reagent cost. (Journal of Biomolecular Screening 2008:1007-1013)
    Creator/Author:
    Rathore, Rakesh; Corr, Jay; Scott, George; Greis, Kenneth D., and Vollmerhaus, Pauline
    Submitter:
    Kenneth Greis
    Date Uploaded:
    03/03/2017
    Date Modified:
    04/07/2017
    Date Created:
    2008-12
    License:
    All rights reserved

  • Development and Validation of a Whole-Cell Inhibition Assay for Bacterial Methionine Aminopeptidase by Surface-Enhanced Laser Desorption Ionization–Time of Flight Mass Spectrometry

    Type:
    Article
    Description/Abstract:
    Bacterial methionine aminopeptidase (MAP) is a protease that removes methionine from the N termini of newly synthesized bacterial proteins after the peptide deformylase enzyme cleaves the formyl group from the initiator formylmethionine. MAP is an essential bacterial gene product and thus represents a potential target for therapeutic intervention. A fundamental challenge in the antibacterial drug discovery field is demonstrating conclusively that compounds with in vitro enzyme inhibition activity produce the desired antibacterial effect by interfering with the same target in whole bacterial cells. One way to address the activity of inhibitor compounds is by profiling cellular biomarkers in whole bacterial cells using compounds that are known inhibitors of a particular target. However, in the case of MAP, no specific inhibitors were available for such studies. Instead, a genetically attenuated MAP strain was generated in which MAP expression was placed under the control of an inducible arabinose promoter. Thus, MAP inhibition in whole cells could be mimicked by growth in the absence of arabinose. This genetically attenuated strain was used as a benchmark for MAP inhibition by profiling whole-cell lysates for unprocessed proteins using surface-enhanced laser desorption ionization–time of flight mass spectrometry (MS). Eight proteins between 4 and 14 kDa were confirmed as being unprocessed and containing the initiator methionine by adding back purified MAP to the preparations prior to MS analysis. Upon establishing these unprocessed proteins as biomarkers for MAP inhibition, the assay was used to screen small-molecule chemical inhibitors of purified MAP for whole-cell activity. Fifteen compound classes yielded three classes of compound with whole-cell activity for further optimization by chemical expansion. This report presents the development, validation, and implementation of a whole-cell inhibition assay for MAP.
    Creator/Author:
    Siehnel, Richard; Howard, Jeremy; Zhou, Songtao; Layh-Schmitt, Gerlinde; Greis, Kenneth D.; Curnow, Alan, and Klanke, Chuck
    Submitter:
    Kenneth Greis
    Date Uploaded:
    03/03/2017
    Date Modified:
    04/07/2017
    Date Created:
    2005-09
    License:
    All rights reserved

  • A ROS-activatable agent elicits homologous recombination DNA repair and synergizes with pathway compounds

    Type:
    Article
    Description/Abstract:
    We have designed ROS-activated cytotoxic agents that are active against AML cancer cells. In this study the mechanism and synergistic effects against cells co-expressing the AML oncogenes MLL-AF9 fusion and FLT3-ITD was investigated. The agent had an IC50 value of 1.8±0.3 μM with a selectivity of 9-fold compared to untransformed cells. Treatment induced DNA strand breaks, apoptosis, and cell cycle arrest. Proteomics and transcriptomics revealed enhanced expression of the pentose phosphate pathway, DNA repair, and pathways common to cell stress. Western blotting confirmed repair by homologous recombination. Importantly, RAC1 treatment was synergistic in combination with multiple pathway targeting therapies in AML cells but less so in untransformed cells. Taken together, these results demonstrate that RAC1 can selectively target poor prognosis AML and do so by creating DNA double strand breaks that require homologous recombination.
    Creator/Author:
    Wunderlich, Mark; Mulloy, James C.; Greis, Kenneth D.; Merino, Edward J.; Thowfeik, Fathima Shazna; Wyder, Michael; AbdulSalam, Safnas F., and Kadekaro, Ana L.
    Submitter:
    Kenneth Greis
    Date Uploaded:
    03/03/2017
    Date Modified:
    04/07/2017
    Date Created:
    2015-09
    License:
    All rights reserved

  • Cardiac myosin binding protein-C is a potential diagnostic biomarker for myocardial infarction

    Type:
    Article
    Description/Abstract:
    Cardiac myosin binding protein-C (cMyBP-C) is a thick filament assembly protein that stabilizes sarcomeric structure and regulates cardiac function; however, the profile of cMyBP-C degradation after myocardial infarction (MI) is unknown. We hypothesized that cMyBP-C is sensitive to proteolysis and is specifically increased in the bloodstream post-MI in rats and humans. Under these circumstances, elevated levels of degraded cMyBP-C could be used as a diagnostic tool to confirm MI. To test this hypothesis, we first established that cMyBP-C dephosphorylation is directly associated with increased degradation of this myofilament protein, leading to its release in vitro. Using neonatal rat ventricular cardiomyocytes in vitro, we were able to correlate the induction of hypoxic stress with increased cMyBP-C dephosphorylation, degradation, and the specific release of N′-fragments. Next, to define the proteolytic pattern of cMyBP-C post-MI, the left anterior descending coronary artery was ligated in adult male rats. Degradation of cMyBP-C was confirmed by a reduction in total cMyBP-C and the presence of degradation products in the infarct tissue. Phosphorylation levels of cMyBP-C were greatly reduced in ischemic areas of the MI heart compared to non-ischemic regions and sham control hearts. Post-MI plasma samples from these rats, as well as humans, were assayed for cMyBP-C and its fragments by sandwich ELISA and immunoprecipitation analyses. Results showed significantly elevated levels of cMyBP-C in the plasma of all post-MI samples. Overall, this study suggests that cMyBP-C is an easily releasable myofilament protein that is dephosphorylated, degraded and released into the circulation post-MI. The presence of elevated levels of cMyBP-C in the blood provides a promising novel biomarker able to accurately rule in MI, thus aiding in the further assessment of ischemic heart disease.
    Creator/Author:
    Muthusamy, Saminathan; Nair, Nandini; McElligott, Andrew; Martin, Jody L.; Govindan, Suresh; Gongora, Enrique; henderson, Kyle K.; Greis, Kenneth D.; Barefield, David; Luther, Pradeep K.; Sadayappan, Sakthivel, and Winegrad, Saul
    Submitter:
    Kenneth Greis
    Date Uploaded:
    03/03/2017
    Date Modified:
    04/07/2017
    Date Created:
    2012-01
    License:
    All rights reserved