[Show abstract][Hide abstract] ABSTRACT: The DNA of all organisms is metabolically active due to persistent endogenous DNA damage, repair and enzyme-mediated base modification pathways important for epigenetic reprogramming and antibody diversity. The free bases released from DNA either spontaneously or by base excision repair pathways constitute DNA metabolites in living tissues. In this study, we have synthesized and characterized the stable-isotope standards for a series of pyrimidines derived from the normal DNA bases by oxidation and deamination. We have used these standards to measure free bases in small molecule extracts from rat brain. Free bases are observed in extracts, consistent with both endogenous DNA damage and 5-methylcytosine demethylation pathways. The most abundant free base observed is uracil, and the potential sources of uracil are discussed. The free bases measured in tissue extracts constitute the end-product of DNA metabolism and could be used to reveal metabolic disturbances in human disease.
Full-text · Article · Oct 2015 · Chemical Research in Toxicology
[Show abstract][Hide abstract] ABSTRACT: Glioblastoma (GBM) is the most common adult primary brain tumor. Despite aggressive multimodal therapy, the survival of patients with GBM remains dismal. However, recent evidence has demonstrated the promise of bone marrow-derived mesenchymal stem cells (BM-hMSCs) as a therapeutic delivery vehicle for anti-glioma agents, due to their ability to migrate or home to human gliomas. While several studies have demonstrated the feasibility of harnessing the homing capacity of BM-hMSCs for targeted delivery of cancer therapeutics, it is now also evident, based on clinically relevant glioma stem cell (GSC) models of GBMs, that BM-hMSCs demonstrate variable tropism towards these tumors. In this study, we compared the lipid environment of GSC xenografts that attract BM-hMSCs (N=9) with those that do not attract (N=9), to identify lipid modalities that are conducive to homing of BM-hMSC to GBMs. We identified lipids directly from tissue by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) and electrospray ionization-tandem mass spectrometry (ESI-MS/MS) of lipid extracts. Several species of signaling lipids, including phosphatidic acid (PA 36:2, PA 40:5, PA 42:5, and PA 42:7) and diacylglycerol (DAG 34:0, DAG 34:1, DAG 36:1, DAG 38:4, DAG 38:6, and DAG 40:6) were lower in attracting xenografts. Molecular lipid images showed that PA (36:2), DAG (40:6), and docosahexaenoic acid (DHA) were decreased within tumor regions of attracting xenografts. Our results provide the first evidence for lipid signaling pathways and lipid-mediated tumor inflammatory responses in the homing of BM-hMSCs to GSC xenografts. Our studies provide new fundamental knowledge on the molecular correlates of the differential homing capacity of BM-hMSCs toward GSC xenografts.
Full-text · Article · Apr 2015 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: We describe the utility of integrated strategies that employ both translation of ENCODE data and major proteomic technology pillars to improve the identification of the “missing proteins”, novel proteoforms, and PTMs. On one hand, databases in combination with bioinformatic tools are efficiently utilized to establish microarray-based transcript analysis and supply rapid protein identifications in clinical samples. On the other hand, sequence libraries are the foundation of targeted protein identification and quantification using mass spectrometric and immunoaffinity techniques. The results from combining proteoENCODEdb searches with experimental mass spectral data indicate that some alternative splicing 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.Keywords: Chromosome-centric Human Protein Project; ENCODE; glioma stem cell; protein sequence mass spectrometry; microassays; missing proteins
No preview · Article · Nov 2014 · Journal of Proteome Research
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
No preview · Article · Jun 2013 · Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
No preview · Article · Dec 2012 · Carbohydrate research
[Show abstract][Hide abstract] 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.
Preview · Article · Feb 2012 · Journal of the American Society for Mass Spectrometry
[Show abstract][Hide abstract] 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.
Full-text · Article · Aug 2011 · Analytical Chemistry
[Show abstract][Hide abstract] 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.
No preview · Article · Aug 2011 · International Journal of Mass Spectrometry
[Show abstract][Hide abstract] 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.
No preview · Article · Jul 2011 · Journal of Proteome Research
[Show abstract][Hide abstract] 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.
No preview · Article · May 2011 · Proceedings of SPIE - The International Society for Optical Engineering
[Show abstract][Hide abstract] 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.
Full-text · Article · Apr 2011 · International Journal of Mass Spectrometry
[Show abstract][Hide abstract] 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.
No preview · Article · Feb 2011 · Canadian Journal of Chemistry
[Show abstract][Hide abstract] ABSTRACT: Intelligent and multitiered quantitative analysis of biological systems rapidly evolves to a key technique in studying biomolecular cancer aspects. Newly emerging advances in both measurement as well as bio-inspired computational techniques have facilitated the development of lipidomics technologies and offer an excellent opportunity to understand regulation at the molecular level in many diseases.
We present computational approaches to study the response of glioblastoma U87 cells to gene- and chemo-therapy. To identify distinct biomarkers and differences in therapeutic outcomes, we develop a novel technique based on graph-clustering. This technique facilitates the exploration and visualization of co-regulations in glioblastoma lipid profiling data. We investigate the changes in the correlation networks for different therapies and study the success of novel gene therapies targeting aggressive glioblastoma.
The novel computational paradigm provides unique "fingerprints" by revealing the intricate interactions at the lipidome level in glioblastoma U87 cells with induced apoptosis (programmed cell death) and thus opens a new window to biomedical frontiers.
Full-text · Article · Sep 2010 · BMC Systems Biology
[Show abstract][Hide abstract] 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.
[Show abstract][Hide abstract] 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.
No preview · Article · Aug 2010 · Rapid Communications in Mass Spectrometry