Mark R Emmett

University of Texas Medical Branch at Galveston, Galveston, TX, United States

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Publications (93)369.77 Total impact

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    ABSTRACT: We describe integrated strategies that employ both translation of ENCODE data and major proteomic technology pillars to improve the identification of the missing proteins, protein isoforms, and PTMs. The results from proteoENCODEdb searches with experimental mass spectral data indicate that some novel splice forms detected at the transcript level are in fact translated to proteins. Our results provide a step toward the directives of the C-HPP initiative and related biomedical research.
    Journal of Proteome Research 11/2014; · 5.06 Impact Factor
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    ABSTRACT: The Chromosome-Centric Human Proteome Project (C-HPP) is a global project aimed to identify at least one protein isoform encoded by the approximately 20, 300 human genes. In addition, protein post-translational modifications will be characterized, with the initial goal of detecting phosphorylation, acetylation, and glycosylation sites in each protein. In this chapter, we provide an overview of known post-translational modifications, their known biological functions, and present strategies to detect them on both a single protein and proteomic scales. In future proteomic studies, global characterization of post-translation modifications, splice variants, and variants caused by single nucleotide polymorphisms (SNPs) will be necessary to fully understand the role of proteins in human biology and disease.
    Genomics and Proteomics for Clinical Discovery and Development, 1 edited by György Marko-Varga, 07/2014: chapter 6: pages 101-136; Springer., ISBN: 978-94-017-9201-1
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    ABSTRACT: While advances in psychotherapy have been made in recent years, drug discovery for brain diseases such as schizophrenia and mood disorders has stagnated. The need for new biomarkers and validated therapeutic targets in the field of neuropsychopharmacology is widely unmet. The brain is the most complex part of human anatomy from the standpoint of number and types of cells, their interconnections and circuitry. To better meet patient needs, improved methods to approach brain studies by understanding functional networks that interact with the genome are being developed. The integrated biological approaches-proteomics, transcriptomics, metabolomics, and glycomics- have a strong record in several areas of biomedicine, including neurochemistry and neuro-oncology. Published applications of an integrated approach to projects of neurological, psychiatric and pharmalogical natures are still few, but show promise to provide deep biological knowledge derived from cells, animal models and clinical materials. Future studes that yield insights based on integrated analyses promise to deliver new therapeutic targets and biomarkers for personalized medicine.Neuropsychopharmacology accepted article preview online, 26 June 2013; doi:10.1038/npp.2013.156.
    Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology 06/2013; · 8.68 Impact Factor
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    ABSTRACT: This paper presents novel graph algorithms and modern control solutions applied to the graph networks resulting from specific experiments to discover disease-related pathways and drug targets in glioma cancer stem cells (GSCs). The theoretical framework applies to many other high-throughput data from experiments relevant to a variety of diseases. In addition to developing novel graph and control networks to predict therapeutic targets, these algorithms will provide biochemists with techniques to identify more metabolic regions and biological pathways for complex diseases, and design and test novel therapeutic solutions.
    SPIE Defense, Security, and Sensing; 05/2013
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    ABSTRACT: Altered carbohydrate metabolism in cancer cells was first noted by Otto Warburg more than 80years ago. Upregulation of genes controlling the glycolytic pathway under normoxia, known as the Warburg effect, clearly differentiates malignant from non-malignant cells. The resurgence of interest in cancer metabolism aims at a better understanding of the metabolic differences between malignant and non-malignant cells and the creation of novel therapeutic and diagnostic agents exploiting these differences. Modified d-glucose and d-mannose analogs were shown to interfere with the metabolism of their respective monosaccharide parent molecules and are potentially clinically useful anticancer and diagnostic agents. One such agent, 2-deoxy-d-glucose (2-DG), has been extensively studied in vitro and in vivo and also clinically evaluated. Studies clearly indicate that 2-DG has a pleiotropic mechanism of action. In addition to effectively inhibiting glycolysis, 2-DG has also been shown to affect protein glycosylation. In order to better understand its molecular mechanism of action, we have designed and synthesized deuterated molecular probes to study 2-DG interference with d-glucose and d-mannose metabolism using mass spectrometry. We present here the synthesis of all desired probes: 2-deutero-d-glucose, 2-deutero-d-mannose, 6-deutero-d-glucose, 6-deutero-d-mannose, and 2-deutero-2-deoxy-d-glucose as well as their complete chemical characterization.
    Carbohydrate research 12/2012; 368C:111-119. · 2.03 Impact Factor
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    ABSTRACT: Hydrogen/deuterium exchange monitored by mass spectrometry is an important non-perturbing tool to study protein structure and protein–protein interactions. However, water in the reversed-phase liquid chromatography mobile phase leads to back-exchange of D for H during chromatographic separation of proteolytic peptides following H/D exchange, resulting in incorrect identification of fast-exchanging hydrogens as unexchanged hydrogens. Previously, fast high-performance liquid chromatography (HPLC) and supercritical fluid chromatography have been shown to decrease back-exchange. Here, we show that replacement of up to 40% of the water in the LC mobile phase by the modifiers, dimethylformamide (DMF) and N-methylpyrrolidone (NMP) (i.e., polar organic modifiers that lack rapid exchanging hydrogens), significantly reduces back-exchange. On-line LC micro-ESI FT-ICR MS resolves overlapped proteolytic peptide isotopic distributions, allowing for quantitative determination of the extent of back-exchange. The DMF modified solvent composition also improves chromatographic separation while reducing back-exchange relative to conventional solvent.
    Journal of the American Society for Mass Spectrometry 02/2012; 23(4):699-707. · 3.59 Impact Factor
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    ABSTRACT: The epitopes of a homohexameric food allergen protein, cashew Ana o 2, identified by two monoclonal antibodies, 2B5 and 1F5, were mapped by solution-phase amide backbone H/D exchange (HDX) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) and the results were compared to previous mapping by immunological and mutational analyses. Antibody 2B5 defines a conformational epitope, and 1F5 defines a linear epitope. Intact murine IgG antibodies were incubated with recombinant Ana o 2 (rAna o 2) to form antigen-monoclonal antibody (Ag-mAb) complexes. mAb-complexed and uncomplexed (free) rAna o 2 were then subjected to HDX. HDX instrumentation and automation were optimized to achieve high sequence coverage by protease XIII digestion. The regions protected from H/D exchange upon antibody binding overlap and thus confirm the previously identified epitope-bearing segments: the first extension of HDX monitored by mass spectrometry to a full-length antigen-antibody complex in solution.
    Analytical Chemistry 08/2011; 83(18):7129-36. · 5.83 Impact Factor
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    ABSTRACT: Natural lipid profiling can improve our current understanding of disease mechanism in a systems biology approach combining genomics, proteomics, and phenotypic changes. However, lipid profiling is complicated by the >10,000 combinations of polar head group, hydrocarbon chain length and degree of unsaturation/hydroxylation, and glycan composition and branching pattern. Here, we show how LC separation coupled with high resolution Fourier transform ion cyclotron resonance mass analysis can quickly narrow down the possible phospholipid and glycosphingolipid compositions. That approach necessitates resolution of mass differences as small as 1.8mDa [12C213C1N1 (51.0064Da) vs. H3O3 (51.0082Da)] in phospholipids and 1.6mDa [13C2S1H2 (59.9944Da) vs. N2O2 (59.9960Da)] in glycosphingolipids. For novel/unknown lipid species, high mass accuracy based Kendrick mass defect analysis enables quick grouping of related lipid species for subsequent tandem MS structural characterization. For sulfur-containing lipid species, high mass resolution can reveal isotopic fine structure to verify assignment.
    International Journal of Mass Spectrometry 08/2011; 305(2):116-119. · 2.23 Impact Factor
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    ABSTRACT: Androgen-repressed human prostate cancer, ARCaP, grows and is highly metastatic to bone and soft tissues in castrated mice. The molecular mechanisms underlying the aberrant responses to androgen are not fully understood. Here, we apply state-of-the-art mass spectrometry methods to investigate the phosphoproteome profiles in ARCaP cells. Because protein biological phosphorylation is always substoichiometric and the ionization efficiency of phosphopeptides is low, selective enrichment of phosphorylated proteins/peptides is required for mass spectrometric analysis of phosphorylation from complex biological samples. Therefore, we compare the sensitivity, efficiency, and specificity for three established enrichment strategies: calcium phosphate precipitation (CPP), immobilized metal ion affinity chromatography (IMAC), and TiO(2)-modified metal oxide chromatography. Calcium phosphate precipitation coupled with the TiO(2) approach offers the best strategy to characterize phosphorylation in ARCaP cells. We analyzed phosphopeptides from ARCaP cells by LC-MS/MS with a hybrid LTQ/FT-ICR mass spectrometer. After database search and stringent filtering, we identified 385 phosphoproteins with an average peptide mass error of 0.32 ± 0.6 ppm. Key identified oncogenic pathways include the mammalian target of rapamycin (mTOR) pathway and the E2F signaling pathway. Androgen-induced proliferation inhibitor (APRIN) was detected in its phosphorylated form, implicating a molecular mechanism underlying the ARCaP phenotype.
    Journal of Proteome Research 07/2011; 10(9):3920-8. · 5.06 Impact Factor
  • Cancer Research 07/2011; 71(8 Supplement):2304-2304. · 9.28 Impact Factor
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    ABSTRACT: Cancer stem cells (CSC) represent a very small percentage of the total tumor population however they pose a big challenge in treating cancer. Glycans play a key role in cancer therapeutics since overexpression of them depending on the glycan type can lead either to cell death or more invasive metastasis. Two major components, fetal bovine serum (FBS) and STAT3, are known to up- or down-regulate certain glycolipid or phospholipid compositions found in glioblastoma CSCs. The analysis and the understanding of the global interactional behavior of lipidomic networks remains a challenging task and can not be accomplished solely based on intuitive reasoning. The present contribution aims at applying graph clustering networks to analyze the functional aspects of certain activators or inhibitors at the molecular level in glioblastoma stem cells (GSCs). This research enhances our understanding of the differences in phenotype changes and determining the responses of glycans to certain treatments for the aggressive GSCs, and represents together with a quantitative phosphoproteomic study1 the most detailed systems biology study of GSCs differentiation known so far. Thus, these new paradigms are providing unique understanding of the mechanisms involved in GSC maintenance and tumorigenicity and are thus opening a new window to biomedical frontiers.
    Proc SPIE 05/2011;
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    ABSTRACT: Cardiac muscle contraction is regulated by the heterotrimeric complex: troponin. We apply solution-phase hydrogen/deuterium exchange monitored by FT-ICR mass spectrometry to study the structural dynamics and the Ca-induced conformational changes of the cardiac isoform of troponin, by comparing H/D exchange rate constants for TnC alone, the binary TnC:TnI complex, and the ternary TnC:TnI:TnT complex for Ca-free and Ca-saturated states. The wide range of exchange rate constants indicates that the complexes possess both highly flexible and very rigid domains. Fast exchange rates were observed for the N-terminal extension of TnI (specific to the cardiac isoform), the DE linker in TnC alone, and the mobile domain of TnI. The slowest rates were for the IT coiled-coil that grants stability and stiffness to the complex. Ca(2+) binding to site II of the N-lobe of TnC induces short-range allosteric effects, mainly protection for the C-lobe of TnC that transmits long-range conformational changes that reach the IT coiled-coil and even TnT1. The present results corroborate prior X-ray crystallography and NMR interpretations and also illuminate domains that were not resolved or truncated in those experiments.
    International Journal of Mass Spectrometry 04/2011; 302(1-3):116-124. · 2.23 Impact Factor
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    ABSTRACT: Petroporphyrin compositional analysis of a heavy crude oil has been realized by isolation and subsequent ESI-FT-ICR mass spectrometric analysis of the porphyrin-containing fractions. Vanadium octaethyl (V=O(II)OEP) and nickel octaethyl (Ni(II)OEP) porphyrin standards were analyzed to determine favorable electrospray ionization conditions and provide insight as to the molecular species present (e.g., adducts, multimers). Standard V=O(II)OEP and Ni(II)OEP solutions revealed the presence of both monomer and dimer species with a greater relative abundance of monomers. In contrast, mass spectral analysis of a porphyrin fraction from Cerro Negro crude oil was dominated by dimeric species. MS3 analysis identified a dioctylphthalate (DOP) contaminant, likely introduced during fractionation of the crude oil. DOP-porphyrin complexes and porphyrin-porphyrin dimers were then identified. Infrared multiphoton dissociation (IRMPD) of dimeric species produced the corresponding monomers with minimal fragmentation. The monomeric petroporphyrins were analyzed to reveal the metal (Ni(II) or V=O(II)), porphyrin type (e.g., etio vs. DPEP), and distribution of alkylation.Key words: petroporphyrin, porphyrin, petroleum, electrospray ionization, mass spectrometry, Fourier transform, ion cyclotron resonance, ICR, FT-ICR, FTMS.
    Canadian Journal of Chemistry 02/2011; 79:546-551. · 1.01 Impact Factor
  • Biophysical Journal 01/2011; 100. · 3.83 Impact Factor
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    ABSTRACT: Electrospray ionization produces multiply charged ions, thereby lowering the mass-to-charge ratio for peptides and small proteins to a range readily accessed by quadrupole ion trap, orbitrap, and ion cyclotron resonance (ICR) mass analyzers (m/z = 400-2000). For Fourier transform mass analyzers (orbitrap and ICR), higher charge also improves signal-to-noise ratio, mass resolution, and mass accuracy. Addition of m-nitrobenzyl alcohol (m-NBA) or sulfolane has previously been shown to increase the charge states of proteins. Moreover, polar aprotic dimethylformamide (DMF) improves chromatographic separation of proteolytic peptides for mass analysis of solution-phase protein hydrogen/deuterium exchange for improved (78-96%) sequence coverage. Here, we show that addition of each of the various modifiers (DMF, thiodiglycol, dimethylacetamide, dimethylsulfoxide, and N-methylpyrrolidone) can significantly increase the charge states of proteins up to 78 kDa. Moreover, incorporation of the same modifiers into reversed-phase liquid chromatography solvents improves sensitivity, charging, and chromatographic resolution for intact proteins.
    Analytical Chemistry 09/2010; 82(17):7515-9. · 5.83 Impact Factor
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    ABSTRACT: Selenomethionine-modified proteins can improve X-ray crystallographic structural resolution by multi-wavelength anomalous diffraction (MAD) phasing. However, the specificity and extent of selenomethionine incorporation must first be assessed. Bottom-up and top-down proteomics with a modified 14.5 T LTQ Fourier transform ion cyclotron resonance mass spectrometer offer a quick, accurate, and robust method to locate and quantify selenomethionine incorporation after auxotrophic expression. Selenomethionine (methionine with sulfur replaced by selenium) has a different natural-abundance isotopic distribution and a mass increase of 47.94 Da relative to wild-type methionine. Here, both wild-type and selenomethionine-substituted forms of the Cas6 protein containing 'clustered regularly interspaced short palindromic repeats' (CRISPRs) were expressed and purified. Comparative bottom-up and top-down proteomics confirmed that all six methionines were fully replaced by selenomethionines in Se-Cas6.
    Rapid Communications in Mass Spectrometry 08/2010; 24(16):2386-92. · 2.51 Impact Factor
  • Xu Wang, Mark R Emmett, Alan G Marshall
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    ABSTRACT: We combine liquid chromatography, electrospray ionization, and Fourier transform ion cyclotron resonance mass spectrometry (LC ESI FT-ICR MS) to determine the sugar composition, linkage pattern, and attachment sites of N-linked glycans. N-linked glycans were enzymatically released from glycoproteins with peptide N-glycosidase F, followed by purification with graphitized carbon cartridge solid-phase extraction and separation over a TSK-Gel Amide80 column under hydrophilic interaction chromatography (HILIC) conditions. Unique glycopeptide compositions were determined from experimentally measured masses for different combinations of glycans and glycopeptides. The method was validated by identifying four peptides glycosylated so as to yield 12 glycopeptides unique in glycan composition for the standard glycoprotein, bovine alpha-2-HS-glycoprotein. We then assigned a total of 137 unique glycopeptide compositions from 18 glycoproteins from fetal bovine serum, and the glycan structures for most of the assigned glycopeptides were heterogeneous. Highly accurate FT-ICR mass measurement is essential for reliable identification.
    Analytical Chemistry 08/2010; 82(15):6542-8. · 5.83 Impact Factor
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    ABSTRACT: Aberrant cell-surface glycosylation patterns are present on virtually all tumors and have been linked to tumor progression, metastasis, and invasivity. We have shown that expressing a normally quiescent, glycoprotein-specific alpha2,6-sialyltransferase (ST6Gal1) gene in gliomas inhibited invasivity in vitro and tumor formation in vivo. To identify other glycogene targets with therapeutic potential, we created a focused 45-mer oligonucleotide microarray platform representing all of the cloned human glycotranscriptome and examined the glycogene expression profiles of 10 normal human brain specimens, 10 malignant gliomas, and 7 human glioma cell lines. Among the many significant changes in glycogene expression observed, of particular interest was the observation that an additional alpha2,6-sialyltransferase, ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminide alpha2,6-sialyltransferase 5 (ST6GalNAcV), was expressed at very low levels in all glioma and glioma cell lines examined compared with normal brain. ST6GalNAcV catalyzes the formation of the terminal alpha2,6-sialic acid linkages on gangliosides. Stable transfection of ST6GalNAcV into U373MG glioma cells produced (i) no change in alpha2,6-linked sialic acid-containing glycoproteins, (ii) increased expression of GM2alpha and GM3 gangliosides and decreased expression of GM1b, Gb3, and Gb4, (iii) marked inhibition of in vitro invasivity, (iv) modified cellular adhesion to fibronectin and laminin, (v) increased adhesion-mediated protein tyrosine phosphorylation of HSPA8, and (vi) inhibition of tumor growth in vivo. These results strongly suggest that modulation of the synthesis of specific glioma cell-surface glycosphingolipids alters invasivity in a manner that may have significant therapeutic potential.
    Proceedings of the National Academy of Sciences 07/2010; 107(28):12646-51. · 9.81 Impact Factor
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    ABSTRACT: Newly emerging advances in both measurement as well as bio-inspired computation techniques have facilitated the development of so-called lipidomics technologies and offer an excellent opportunity to understand regulation at the molecular level in many diseases such as cancer. The analysis and the understanding of the global interactional behavior of lipidomic networks remains a challenging task and can not be accomplished solely based on intuitive reasoning. The present contribution aims at developing novel computational approaches to assess the topological and functional aspects of lipidomic networks and discusses their benefits compared to recently proposed techniques. Graph-clustering methods are introduced as powerful correlation networks which enable a simultaneous exploration and visualization of co-regulation in glioblastoma data. The dynamic description of the lipidomic network is given through multi-mode nonlinear autonomous stochastic systems to model the interactions at the molecular level and to study the success of novel gene therapies for eradicating the aggressive glioblastoma. These new paradigms are providing unique "fingerprints" by revealing how the intricate interactions at the lipidome level can be employed to induce apoptosis (cell death) and are thus opening a new window to biomedical frontiers.© (2010) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.
    04/2010;
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    ABSTRACT: Mutations of the receptor tyrosine kinase KIT are linked to certain cancers such as gastrointestinal stromal tumors (GISTs). Biophysical, biochemical, and structural studies have provided insight into the molecular basis of resistance to the KIT inhibitors, imatinib and sunitinib. Here, solution-phase hydrogen/deuterium exchange (HDX) and direct binding mass spectrometry experiments provide a link between static structure models and the dynamic equilibrium of the multiple states of KIT, supporting that sunitinib targets the autoinhibited conformation of WT-KIT. The D816H mutation shifts the KIT conformational equilibrium toward the activated state. The V560D mutant exhibits two low energy conformations: one is more flexible and resembles the D816H mutant shifted toward the activated conformation, and the other is less flexible and resembles the wild-type KIT in the autoinhibited conformation. This result correlates with the V560D mutant exhibiting a sensitivity to sunitinib that is less than for WT KIT but greater than for KIT D816H. These findings support the elucidation of the resistance mechanism for the KIT mutants.
    Protein Science 04/2010; 19(4):703-15. · 2.86 Impact Factor

Publication Stats

3k Citations
369.77 Total Impact Points

Institutions

  • 2012–2013
    • University of Texas Medical Branch at Galveston
      • Department of Biochemistry and Molecular Biology
      Galveston, TX, United States
  • 1993–2012
    • Florida State University
      • Department of Chemistry and Biochemistry
      Tallahassee, FL, United States
  • 2010
    • Karlsruhe Institute of Technology
      Carlsruhe, Baden-Württemberg, Germany
  • 2007–2010
    • National High Magnetic Field Laboratory
      Tallahassee, Florida, United States
    • École Polytechnique Fédérale de Lausanne
      • Laboratoire de spectrométrie de masse de biomolécules
      Lausanne, VD, Switzerland
  • 2005
    • Valdosta State University
      • Department of Chemistry
      Valdosta, GA, United States
  • 2003–2004
    • University of Alabama at Birmingham
      • Department of Microbiology
      Birmingham, AL, United States
    • Eastern Idaho Regional Medical Center
      Idaho Falls, Idaho, United States
  • 1994–1995
    • University of Texas Medical School
      • Department of Biochemistry and Molecular Biology
      Houston, Texas, United States