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1
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Carbonara K, Padula MP, Coorssen JR. Quantitative assessment confirms deep proteome analysis by integrative top-down proteomics. Electrophoresis 2023; 44:472-480. [PMID: 36416355 DOI: 10.1002/elps.202200257] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2022] [Revised: 11/17/2022] [Accepted: 11/18/2022] [Indexed: 11/24/2022]
Abstract
The goal of integrative top-down proteomics (i.e., two-dimensional gel electrophoresis [2DE] coupled with liquid chromatography and tandem mass spectrometry [LC/MS/MS]) is a routine analytical approach that fully addresses the breadth and depth of proteomes. To accomplish this, there should be no addition, removal, or modification to any constituent proteoforms. To address two-decade old claims of protein losses during front-end proteome resolution using 2DE, here we tested an alternate rehydration method for immobilized pH gradient strips prior to isoelectric focusing (IEF; i.e., faceup compared to facedown) and quantitatively assessed losses during the front-end of 2DE (rehydration and IEF). Using a well-established high-resolution, quantitative 2DE protocol, there were no detectable proteoform losses using the alternate faceup rehydration method. Although there is a <0.25% total loss of proteoforms during standard facedown rehydration, it is insignificant in terms of having any effect on overall proteome resolution (i.e., total spot count and total spot signal). This report is another milestone in integrative top-down proteomics, disproving long-held dogma in the field and confirming that quantitative front-end 2DE/LC/MS/MS is currently the only method to broadly and deeply analyze proteomes by resolving their constituent proteoforms.
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Affiliation(s)
- Katrina Carbonara
- Department of Health Sciences, Faculty of Applied Health Sciences, Brock University, St. Catharines, Ontario, Canada
| | - Matthew P Padula
- School of Life Sciences and Proteomics, Lipidomics and Metabolomics Core Facility, Faculty of Science, University of Technology Sydney, Sydney, New South Wales, Australia
| | - Jens R Coorssen
- Department of Biological Sciences, Faculty of Mathematics and Science, Brock University, St. Catharines, Ontario, Canada.,Ronin Institute, Montclair, New Jersey, USA
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2
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Carbonara K, Coorssen JR. Sometimes faster can be better: Microneedling IPG strips enables higher throughput for integrative top-down proteomics. Proteomics 2023; 23:e2200307. [PMID: 36349823 DOI: 10.1002/pmic.202200307] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2022] [Revised: 09/27/2022] [Accepted: 11/02/2022] [Indexed: 11/10/2022]
Abstract
Passive rehydration of immobilized pH gradient (IPG) strips for two-dimensional gel electrophoresis (2DE) has, to our knowledge, never been quantitatively evaluated to determine an ideal rehydration time. Seeking to increase throughput without sacrificing analytical rigor, we report that a substantially shorter rehydration time is accomplished when surface area of IPG strips is increased via microneedling. Rehydration for 4 h, post microneedling, provides comparable results to overnight rehydration in final analyses by 2DE, while also shortening the overall protocol by 1 day.
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Affiliation(s)
- Katrina Carbonara
- Department of Health Sciences, Faculty of Applied Health Sciences, Brock University, St. Catharines, Ontario, Canada
| | - Jens R Coorssen
- Department of Biological Sciences, Faculty of Mathematics and Science, Brock University, St. Catharines, Ontario, Canada.,Ronin Institute, Montclair, New Jersey, USA
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3
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May C, Brosseron F, Pfeiffer K, Fuchs K, Meyer HE, Sitek B, Marcus K. Proteome Analysis with Classical 2D-PAGE. Methods Mol Biol 2021; 2228:53-62. [PMID: 33950483 DOI: 10.1007/978-1-0716-1024-4_5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is based on the combination of two orthogonal separation techniques. In the first dimension, proteins are separated by their isoelectric point, a technique known as isoelectric focusing (IEF). There are two important variants of IEF, which are carrier-ampholine (CA)-based IEF and immobilized pH-gradient (IPG)-based IEF. In the second dimension, proteins are further separated by their electrophoretic mobility using SDS-PAGE. Finally, proteins can be visualized and quantified by different staining procedures such as Coomassie, silver staining, or fluorescence labeling. This article gives detailed protocols for 2D-PAGE, using both CA- and IPG-based separation in the first dimension.
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Affiliation(s)
- Caroline May
- Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany
- Medical Proteome Analysis, Center for Proteindiagnostics (PRODI) Ruhr-University Bochum, Bochum, Germany
| | - Frederic Brosseron
- Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany
| | - Kathy Pfeiffer
- Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany
- Medical Proteome Analysis, Center for Proteindiagnostics (PRODI) Ruhr-University Bochum, Bochum, Germany
| | - Kristin Fuchs
- Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany
- Medical Proteome Analysis, Center for Proteindiagnostics (PRODI) Ruhr-University Bochum, Bochum, Germany
- Klinik für Anästhesiologie, Intensivmedizin und Schmerztherapie, Universitätsklinikum Knappschaftskrankenhaus Bochum, Bochum, Germany
| | - Helmut E Meyer
- Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany
| | - Barbara Sitek
- Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany
- Medical Proteome Analysis, Center for Proteindiagnostics (PRODI) Ruhr-University Bochum, Bochum, Germany
- Klinik für Anästhesiologie, Intensivmedizin und Schmerztherapie, Universitätsklinikum Knappschaftskrankenhaus Bochum, Bochum, Germany
| | - Katrin Marcus
- Medizinisches Proteom-Center (MPC), Medical Faculty, Ruhr-University Bochum, Bochum, Germany.
- Medical Proteome Analysis, Center for Proteindiagnostics (PRODI) Ruhr-University Bochum, Bochum, Germany.
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Anti-PDHA1 antibody is detected in a subset of patients with schizophrenia. Sci Rep 2020; 10:7906. [PMID: 32404964 PMCID: PMC7220915 DOI: 10.1038/s41598-020-63776-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2020] [Accepted: 04/06/2020] [Indexed: 11/30/2022] Open
Abstract
Autoantibodies have been implicated in schizophrenia aetiology. Here, novel autoantibodies were isolated from patients with schizophrenia. Autoantibody candidates were searched using two-dimensional gel electrophoresis and western blotting with rat brain proteins as antigens and two sera pools (25 schizophrenia patients versus 25 controls) as antibodies. Immunoreactive antigens were identified by mass spectrometry. Antibody prevalence were evaluated by western blotting using human recombinant proteins. Furthermore, brain magnetic resonance imaging data (regional brain volumes and diffusion tensor imaging measures) were compared. Two proteins of the mitochondrial respiration pathway were identified as candidate antigens. Three patients with schizophrenia, but no controls, expressed antibodies targeting one of the candidate antigens, i.e., pyruvate dehydrogenase E1 component subunit alpha, somatic form, mitochondrial (PDHA1, EC 1.2.4.1), which is related to mitochondrial energy production. Anti-PDHA1 antibody-positive patients (n = 3) had increased volumes in the left occipital fusiform gyrus compared to both controls (n = 23, p = 0.017) and antibody-negative patients (n = 16, p = 0.009), as well as in the left cuneus compared to antibody-negative patients (n = 16, p = 0.018). This is the first report of an anti-PDHA1 antibody in patients with schizophrenia. Compatible with recent findings of mitochondrial dysfunction in schizophrenia, this antibody may be involved in the pathogenesis of a specific subgroup of schizophrenia.
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Hashimoto A, Takeuchi S, Kajita R, Yamagata A, Kakui R, Tanaka T, Nakata K. Proteogenomic analysis of granulocyte macrophage colony- stimulating factor autoantibodies in the blood of a patient with autoimmune pulmonary alveolar proteinosis. Sci Rep 2020; 10:4923. [PMID: 32188922 PMCID: PMC7080758 DOI: 10.1038/s41598-020-61934-y] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2019] [Accepted: 03/02/2020] [Indexed: 11/18/2022] Open
Abstract
Recently, attempts to reveal the structures of autoantibodies comprehensively using improved proteogenomics technology, have become popular. This technology identifies peptides in highly purified antibodies by using an Orbitrap device to compare spectra from liquid chromatography-tandem mass spectrometry against a cDNA database obtained through next-generation sequencing. In this study, we first analyzed granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibodies in a patient with autoimmune pulmonary alveolar proteinosis, using the trapped ion mobility spectrometry coupled with quadrupole time-of-flight (TIMS-TOF) instrument. The TIMS-TOF instrument identified peptides that partially matched sequences in up to 156 out of 162 cDNA clones. Complementarity-determining region 3 (CDR3) was fully and partially detected in nine and 132 clones, respectively. Moreover, we confirmed one unique framework region 4 (FR4) and at least three unique across CDR3 to FR4 peptides via de novo peptide sequencing. This new technology may thus permit the comprehensive identification of autoantibody structure.
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Affiliation(s)
| | - Shiho Takeuchi
- Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
| | | | | | | | - Takahiro Tanaka
- Niigata University Medical & Dental Hospital, Niigata, Japan
| | - Koh Nakata
- Niigata University Medical & Dental Hospital, Niigata, Japan.
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Dalzon B, Bons J, Diemer H, Collin-Faure V, Marie-Desvergne C, Dubosson M, Cianferani S, Carapito C, Rabilloud T. A Proteomic View of Cellular Responses to Anticancer Quinoline-Copper Complexes. Proteomes 2019; 7:26. [PMID: 31238524 PMCID: PMC6630412 DOI: 10.3390/proteomes7020026] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/28/2019] [Revised: 06/18/2019] [Accepted: 06/21/2019] [Indexed: 02/07/2023] Open
Abstract
Metal-containing drugs have long been used in anticancer therapies. The mechansims of action of platinum-based drugs are now well-understood, which cannot be said of drugs containing other metals, such as gold or copper. To gain further insights into such mechanisms, we used a classical proteomic approach based on two-dimensional elelctrophoresis to investigate the mechanisms of action of a hydroxyquinoline-copper complex, which shows promising anticancer activities, using the leukemic cell line RAW264.7 as the biological target. Pathway analysis of the modulated proteins highlighted changes in the ubiquitin/proteasome pathway, the mitochondrion, the cell adhesion-cytoskeleton pathway, and carbon metabolism or oxido-reduction. In line with these prteomic-derived hypotheses, targeted validation experiments showed that the hydroxyquinoline-copper complex induces a massive reduction in free glutathione and a strong alteration in the actin cytoskeleton, suggesting a multi-target action of the hydroxyquinoline-copper complex on cancer cells.
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Affiliation(s)
- Bastien Dalzon
- Chemistry and Biology of Metals, Univ. Grenoble Alpes, CNRS UMR5249, CEA, IRIG,CBM, F-38054 Grenoble, France.
| | - Joanna Bons
- Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), Université de Strasbourg, CNRS, IPHC UMR 7178, 67000 Strasbourg, France.
| | - Hélène Diemer
- Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), Université de Strasbourg, CNRS, IPHC UMR 7178, 67000 Strasbourg, France.
| | - Véronique Collin-Faure
- Chemistry and Biology of Metals, Univ. Grenoble Alpes, CNRS UMR5249, CEA, IRIG,CBM, F-38054 Grenoble, France.
| | - Caroline Marie-Desvergne
- Nanosafety Platform, Medical Biology Laboratory (LBM), Univ. Grenoble-Alpes, CEA, 17 rue des Martyrs, F-38054 Grenoble, France.
| | - Muriel Dubosson
- Nanosafety Platform, Medical Biology Laboratory (LBM), Univ. Grenoble-Alpes, CEA, 17 rue des Martyrs, F-38054 Grenoble, France.
| | - Sarah Cianferani
- Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), Université de Strasbourg, CNRS, IPHC UMR 7178, 67000 Strasbourg, France.
| | - Christine Carapito
- Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), Université de Strasbourg, CNRS, IPHC UMR 7178, 67000 Strasbourg, France.
| | - Thierry Rabilloud
- Chemistry and Biology of Metals, Univ. Grenoble Alpes, CNRS UMR5249, CEA, IRIG,CBM, F-38054 Grenoble, France.
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A quantitative proteomic analysis of cofilin phosphorylation in myeloid cells and its modulation using the LIM kinase inhibitor Pyr1. PLoS One 2018; 13:e0208979. [PMID: 30550596 PMCID: PMC6294390 DOI: 10.1371/journal.pone.0208979] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2018] [Accepted: 11/28/2018] [Indexed: 01/10/2023] Open
Abstract
LIM kinases are located at a strategic crossroad, downstream of several signaling pathways and upstream of effectors such as microtubules and the actin cytoskeleton. Cofilin is the only LIM kinases substrate that is well described to date, and its phosphorylation on serine 3 by LIM kinases controls cofilin actin-severing activity. Consequently, LIM kinases inhibition leads to actin cytoskeleton disorganization and blockade of cell motility, which makes this strategy attractive in anticancer treatments. LIMK has also been reported to be involved in pathways that are deregulated in hematologic malignancies, with little information regarding cofilin phosphorylation status. We have used proteomic approaches to investigate quantitatively and in detail the phosphorylation status of cofilin in myeloid tumor cell lines of murine and human origin. Our results show that under standard conditions, only a small fraction (10 to 30% depending on the cell line) of cofilin is phosphorylated (including serine 3 phosphorylation). In addition, after a pharmacological inhibition of LIM kinases, a residual cofilin phosphorylation is observed on serine 3. Interestingly, this 2D gel based proteomic study identified new phosphorylation sites on cofilin, such as threonine 63, tyrosine 82 and serine 108.
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Kashiwazaki D, Uchino H, Kuroda S. Downregulation of Apolipoprotein-E and Apolipoprotein-J in Moyamoya Disease-A Proteome Analysis of Cerebrospinal Fluid. J Stroke Cerebrovasc Dis 2017; 26:2981-2987. [PMID: 28843803 DOI: 10.1016/j.jstrokecerebrovasdis.2017.07.028] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2017] [Revised: 07/18/2017] [Accepted: 07/25/2017] [Indexed: 01/09/2023] Open
Abstract
BACKGROUND AND PURPOSE Genetic factors are closely involved in the etiology of moyamoya disease (MMD). However, its postgenomic mechanisms are still unknown. This study was aimed to identify specific biomarkers in the cerebrospinal fluid (CSF) of patients with MMD, using quantitative proteome technique. METHODS This study included 10 patients with MMD and 4 controls. The CSF was collected without blood contamination during surgery. A comparative 2-dimensional gel electrophoresis study (2D-PAGE) was performed. Protein spots that showed significant differences between moyamoya patients and controls were selected for further analysis by mass spectrometry. RESULTS On 2D-PAGE, 2 proteins were significantly upregulated, and 2 other proteins were downregulated in the CSF of MMD. Further mass spectrometry analysis revealed that haptoglobin and α-1-B-glycoprotein (A1BG) were upregulated. On the other hand, apolipoprotein-E (apoE), apoE precursor, and apolipoprotein-J (apoJ) were significantly downregulated in the CSF of MMD. The observed probability-based MOWSE score was 72 for haptoglobin (P <.05), 521 for A1BG (P <.05), 62 for apoE (P <.05), 72 for apoE precursor (P <.05), and 112 for apoJ (P <.05). CONCLUSION Although the role of A1BG in the central nervous system is still unknown, the overexpressed haptoglobin may indicate the inflammation and/or angiogenesis in MMD. The downregulation of apoE and apoJ strongly suggests a critical role of lipid metabolism in the development and progression of MMD. These proteins may be novel biomarkers in shedding light on the pathogenesis of MMD, although further studies would be warranted.
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Affiliation(s)
- Daina Kashiwazaki
- Department of Neurosurgery, Graduate School of Medicine and Pharmaceutical Science, University of Toyama University, Toyama, Japan.
| | - Haruto Uchino
- Department of Neurosurgery, Graduate School of Medicine and Pharmaceutical Science, University of Toyama University, Toyama, Japan
| | - Satoshi Kuroda
- Department of Neurosurgery, Graduate School of Medicine and Pharmaceutical Science, University of Toyama University, Toyama, Japan
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Dalzon B, Aude-Garcia C, Collin-Faure V, Diemer H, Béal D, Dussert F, Fenel D, Schoehn G, Cianférani S, Carrière M, Rabilloud T. Differential proteomics highlights macrophage-specific responses to amorphous silica nanoparticles. NANOSCALE 2017; 9:9641-9658. [PMID: 28671223 DOI: 10.1039/c7nr02140b] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/23/2023]
Abstract
The technological and economic benefits of engineered nanomaterials may be offset by their adverse effects on living organisms. One of the highly produced nanomaterials under such scrutiny is amorphous silica nanoparticles, which are known to have an appreciable, although reversible, inflammatory potential. This is due to their selective toxicity toward macrophages, and it is thus important to study the cellular responses of this cell type to silica nanoparticles to better understand the direct or indirect adverse effects of nanosilica. We have here studied the responses of the RAW264.7 murine macrophage cells and of the control MPC11 plasma cells to subtoxic concentrations of nanosilica, using a combination of proteomic and targeted approaches. This allowed us to document alterations in the cellular cytoskeleton, in the phagocytic capacity of the cells as well as their ability to respond to bacterial stimuli. More surprisingly, silica nanoparticles also induce a greater sensitivity of macrophages to DNA alkylating agents, such as styrene oxide, even at doses which do not induce any appreciable cell death.
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Affiliation(s)
- Bastien Dalzon
- Laboratory of Chemistry and Biology of Metals, UMR 5249, Univ. Grenoble Alpes, CNRS, CEA, Grenoble, France.
| | - Catherine Aude-Garcia
- Laboratory of Chemistry and Biology of Metals, UMR 5249, Univ. Grenoble Alpes, CNRS, CEA, Grenoble, France.
| | - Véronique Collin-Faure
- Laboratory of Chemistry and Biology of Metals, UMR 5249, Univ. Grenoble Alpes, CNRS, CEA, Grenoble, France.
| | - Hélène Diemer
- Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS, IPHC UMR 7178, 67000 Strasbourg, France
| | - David Béal
- Chimie Interface Biologie pour l'Environnement, la Santé et la Toxicologie (CIBEST), UMR 5819, Univ. Grenoble Alpes, CEA, CNRS, INAC, SyMMES, F-38000 Grenoble, France
| | - Fanny Dussert
- Chimie Interface Biologie pour l'Environnement, la Santé et la Toxicologie (CIBEST), UMR 5819, Univ. Grenoble Alpes, CEA, CNRS, INAC, SyMMES, F-38000 Grenoble, France
| | - Daphna Fenel
- Institut de Biologie Structurale Jean-Pierre Ebel, UMR5075, Univ. Grenoble Alpes, CEA, CNRS, Grenoble, France
| | - Guy Schoehn
- Institut de Biologie Structurale Jean-Pierre Ebel, UMR5075, Univ. Grenoble Alpes, CEA, CNRS, Grenoble, France
| | - Sarah Cianférani
- Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS, IPHC UMR 7178, 67000 Strasbourg, France
| | - Marie Carrière
- Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS, IPHC UMR 7178, 67000 Strasbourg, France
| | - Thierry Rabilloud
- Laboratory of Chemistry and Biology of Metals, UMR 5249, Univ. Grenoble Alpes, CNRS, CEA, Grenoble, France.
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Dalzon B, Diemer H, Collin-Faure V, Cianférani S, Rabilloud T, Aude-Garcia C. Culture medium associated changes in the core proteome of macrophages and in their responses to copper oxide nanoparticles. Proteomics 2016; 16:2864-2877. [DOI: 10.1002/pmic.201600052] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2016] [Revised: 09/15/2016] [Accepted: 10/06/2016] [Indexed: 11/10/2022]
Affiliation(s)
- Bastien Dalzon
- Laboratory of Chemistry and Biology of Metals; BIG/CBM, CEA Grenoble; Grenoble France
- Laboratory of Chemistry and Biology of Metals; University of Grenoble Alpes; Grenoble France
- Laboratory of Chemistry and Biology of Metals; CNRS UMR5249 Grenoble France
| | - Hélène Diemer
- BioOrganic Mass Spectrometry Laboratory (LSMBO); IPHC, Université de Strasbourg; Strasbourg France
- BioOrganic Mass Spectrometry Laboratory (LSMBO); CNRS UMR7178 Strasbourg France
| | - Véronique Collin-Faure
- Laboratory of Chemistry and Biology of Metals; BIG/CBM, CEA Grenoble; Grenoble France
- Laboratory of Chemistry and Biology of Metals; University of Grenoble Alpes; Grenoble France
- Laboratory of Chemistry and Biology of Metals; CNRS UMR5249 Grenoble France
| | - Sarah Cianférani
- BioOrganic Mass Spectrometry Laboratory (LSMBO); IPHC, Université de Strasbourg; Strasbourg France
- BioOrganic Mass Spectrometry Laboratory (LSMBO); CNRS UMR7178 Strasbourg France
| | - Thierry Rabilloud
- Laboratory of Chemistry and Biology of Metals; BIG/CBM, CEA Grenoble; Grenoble France
- Laboratory of Chemistry and Biology of Metals; University of Grenoble Alpes; Grenoble France
- Laboratory of Chemistry and Biology of Metals; CNRS UMR5249 Grenoble France
| | - Catherine Aude-Garcia
- Laboratory of Chemistry and Biology of Metals; BIG/CBM, CEA Grenoble; Grenoble France
- Laboratory of Chemistry and Biology of Metals; University of Grenoble Alpes; Grenoble France
- Laboratory of Chemistry and Biology of Metals; CNRS UMR5249 Grenoble France
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Aude-Garcia C, Dalzon B, Ravanat JL, Collin-Faure V, Diemer H, Strub JM, Cianferani S, Van Dorsselaer A, Carrière M, Rabilloud T. A combined proteomic and targeted analysis unravels new toxic mechanisms for zinc oxide nanoparticles in macrophages. J Proteomics 2016; 134:174-185. [DOI: 10.1016/j.jprot.2015.12.013] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2015] [Revised: 11/16/2015] [Accepted: 12/09/2015] [Indexed: 12/21/2022]
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de Oliveira Souza VC, de Marco KC, Laure HJ, Rosa JC, Barbosa F. A brain proteome profile in rats exposed to methylmercury or thimerosal (ethylmercury). JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH. PART A 2016; 79:502-512. [PMID: 27294299 DOI: 10.1080/15287394.2016.1182003] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/06/2023]
Abstract
Exposure to organomercurials has been associated with harmful effects on the central nervous system (CNS). However, the mechanisms underlying organomercurial-mediated neurotoxic effects need to be elucidated. Exposure to toxic elements may promote cellular modifications such as alterations in protein synthesis in an attempt to protect tissues and organs from damage. In this context, the use of a "proteomic profile" is an important tool to identify potential early biomarkers or targets indicative of neurotoxicity. The aim of this study was to investigate potential modifications in rat cerebral cell proteome following exposure to methylmercury (MeHg) or ethylmercury (EtHg). For MeHg exposure, animals were administered by gavage daily 140 µg/kg/d of Hg (as MeHg) for 60 d and sacrificed 24 h after the last treatment. For EtHg exposure, 800 µg/kg/d of Hg (as EtHg) was given intramuscularly (im) in a single dose and rats were sacrificed after 4 h. Control groups received saline either by gavage or im. After extraction of proteins from whole brain samples and separation by two-dimensional electrophoresis (2-DE), 26 differentially expressed proteins were identified from exposed animals by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF). Both MeHg and EtHg exposure induced an overexpression of calbindin, a protein that acts as a neuroprotective agent by (1) adjusting the concentration of Ca(2+) within cells and preventing neurodegenerative diseases and (2) decreasing expression of glutamine synthetase, a crucial protein involved in regulation of glutamate concentration in synaptic cleft. In contrast, expression of superoxide dismutase (SOD), a protein involved in antioxidant defense, was elevated in brain of MeHg-exposed animals. Taken together, our data provide new valuable information on the possible molecular mechanisms associated with MeHg- and EtHg-mediated toxicity in cerebral tissue. These observed protein alterations may be considered as biomarkers candidates for biological monitoring of organomercurial poisoning.
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Affiliation(s)
- Vanessa Cristina de Oliveira Souza
- a Department of Clinical, Bromatological and Toxicological Analysis, Faculty of Pharmaceutical Sciences of Ribeirão Preto , University of São Paulo , São Paulo , Brazil
| | - Kátia Cristina de Marco
- a Department of Clinical, Bromatological and Toxicological Analysis, Faculty of Pharmaceutical Sciences of Ribeirão Preto , University of São Paulo , São Paulo , Brazil
| | - Hélen Julie Laure
- b Department of Molecular and Cellular Biology, Faculty of Medicine of Ribeirão Preto , University of São Paulo , São Paulo , Brazil
| | - José Cesar Rosa
- b Department of Molecular and Cellular Biology, Faculty of Medicine of Ribeirão Preto , University of São Paulo , São Paulo , Brazil
| | - Fernando Barbosa
- a Department of Clinical, Bromatological and Toxicological Analysis, Faculty of Pharmaceutical Sciences of Ribeirão Preto , University of São Paulo , São Paulo , Brazil
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Nucleic acid import into mitochondria: New insights into the translocation pathways. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2015; 1853:3165-81. [DOI: 10.1016/j.bbamcr.2015.09.011] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/29/2015] [Revised: 08/16/2015] [Accepted: 09/10/2015] [Indexed: 11/18/2022]
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14
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Gorasia DG, Veith PD, Chen D, Seers CA, Mitchell HA, Chen YY, Glew MD, Dashper SG, Reynolds EC. Porphyromonas gingivalis Type IX Secretion Substrates Are Cleaved and Modified by a Sortase-Like Mechanism. PLoS Pathog 2015; 11:e1005152. [PMID: 26340749 PMCID: PMC4560394 DOI: 10.1371/journal.ppat.1005152] [Citation(s) in RCA: 78] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2015] [Accepted: 08/14/2015] [Indexed: 12/11/2022] Open
Abstract
The type IX secretion system (T9SS) of Porphyromonas gingivalis secretes proteins possessing a conserved C-terminal domain (CTD) to the cell surface. The C-terminal signal is essential for these proteins to translocate across the outer membrane via the T9SS. On the surface the CTD of these proteins is cleaved prior to extensive glycosylation. It is believed that the modification on these CTD proteins is anionic lipopolysaccharide (A-LPS), which enables the attachment of CTD proteins to the cell surface. However, the exact site of modification and the mechanism of attachment of CTD proteins to the cell surface are unknown. In this study we characterized two wbaP (PG1964) mutants that did not synthesise A-LPS and accumulated CTD proteins in the clarified culture fluid (CCF). The CTDs of the CTD proteins in the CCF were cleaved suggesting normal secretion, however, the CTD proteins were not glycosylated. Mass spectrometric analysis of CTD proteins purified from the CCF of the wbaP mutants revealed the presence of various peptide/amino acid modifications from the growth medium at the C-terminus of the mature CTD proteins. This suggested that modification occurs at the C-terminus of T9SS substrates in the wild type P. gingivalis. This was confirmed by analysis of CTD proteins from wild type, where a 648 Da linker was identified to be attached at the C-terminus of mature CTD proteins. Importantly, treatment with proteinase K released the 648 Da linker from the CTD proteins demonstrating a peptide bond between the C-terminus and the modification. Together, this is suggestive of a mechanism similar to sortase A for the cleavage and modification/attachment of CTD proteins in P. gingivalis. PG0026 has been recognized as the CTD signal peptidase and is now proposed to be the sortase-like protein in P. gingivalis. To our knowledge, this is the first biochemical evidence suggesting a sortase-like mechanism in Gram-negative bacteria.
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Affiliation(s)
- Dhana G. Gorasia
- Oral Health Cooperative Research Centre, Melbourne Dental School, and The Bio21 Institute, The University of Melbourne, Victoria, Australia
| | - Paul D. Veith
- Oral Health Cooperative Research Centre, Melbourne Dental School, and The Bio21 Institute, The University of Melbourne, Victoria, Australia
| | - Dina Chen
- Oral Health Cooperative Research Centre, Melbourne Dental School, and The Bio21 Institute, The University of Melbourne, Victoria, Australia
| | - Christine A. Seers
- Oral Health Cooperative Research Centre, Melbourne Dental School, and The Bio21 Institute, The University of Melbourne, Victoria, Australia
| | - Helen A. Mitchell
- Oral Health Cooperative Research Centre, Melbourne Dental School, and The Bio21 Institute, The University of Melbourne, Victoria, Australia
| | - Yu-Yen Chen
- Oral Health Cooperative Research Centre, Melbourne Dental School, and The Bio21 Institute, The University of Melbourne, Victoria, Australia
| | - Michelle D. Glew
- Oral Health Cooperative Research Centre, Melbourne Dental School, and The Bio21 Institute, The University of Melbourne, Victoria, Australia
| | - Stuart G. Dashper
- Oral Health Cooperative Research Centre, Melbourne Dental School, and The Bio21 Institute, The University of Melbourne, Victoria, Australia
| | - Eric C. Reynolds
- Oral Health Cooperative Research Centre, Melbourne Dental School, and The Bio21 Institute, The University of Melbourne, Victoria, Australia
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15
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Armand L, Biola-Clier M, Bobyk L, Collin-Faure V, Diemer H, Strub JM, Cianferani S, Van Dorsselaer A, Herlin-Boime N, Rabilloud T, Carriere M. Molecular responses of alveolar epithelial A549 cells to chronic exposure to titanium dioxide nanoparticles: A proteomic view. J Proteomics 2015; 134:163-173. [PMID: 26276045 DOI: 10.1016/j.jprot.2015.08.006] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2015] [Revised: 08/05/2015] [Accepted: 08/07/2015] [Indexed: 12/22/2022]
Abstract
UNLABELLED Although the biological effects of titanium dioxide nanoparticles (TiO2-NPs) have been studied for more than two decades, the mechanisms governing their toxicity are still unclear. We applied 2D-gel proteomics analysis on A549 epithelial alveolar cells chronically exposed for 2months to 2.5 or 50μg/mL of deeply characterized TiO2-NPs, in order to obtain comprehensive molecular responses that may reflect functional outcomes. We show that exposure to TiO2-NPs impacts the abundance of 30 protein species, corresponding to 22 gene products. These proteins are involved in glucose metabolism, trafficking, gene expression, mitochondrial function, proteasome activity and DNA damage response. Besides, our results suggest that p53 pathway is activated, slowing down cell cycle progression and reducing cell proliferation rate. Moreover, we report increased content of chaperones-related proteins, which suggests homeostasis re-establishment. Finally, our results highlight that chronic exposure to TiO2-NPs affects the same cellular functions as acute exposure to TiO2-NPs, although lower exposure concentrations and longer exposure times induce more intense cellular response. BIOLOGICAL SIGNIFICANCE Our results make possible the identification of new mechanisms that explain TiO2-NP toxicity upon long-term, in vitro exposure of A549 cells. It is the first article describing -omics results obtained with this experimental strategy. We show that this long-term exposure modifies the cellular content of proteins involved in functions including mitochondrial activity, intra- and extracellular trafficking, proteasome activity, glucose metabolism, and gene expression. Moreover we observe modification of content of proteins that activate the p53 pathway, which suggest the induction of a DNA damage response. Technically, our results show that exposure of A549 cells to a high concentration of TiO2-NPs leads to the identification of modulations of the same functional categories than exposure to low, more realistic concentrations. Still the intensity differs between these two exposure scenarios. We also show that chronic exposure to TiO2-NPs induces the modulation of cellular functions that have already been reported in the literature as being impacted in acute exposure scenarios. This proves that the exposure protocol in in vitro experiments related to nanoparticle toxicology might be cautiously chosen since inappropriate scenario may lead to inappropriate and/or incomplete conclusions.
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Affiliation(s)
- Lucie Armand
- Université Grenoble-Alpes, INAC-LCIB, Laboratoire Lésions des Acides Nucléiques, 17 rue des Martyrs, F-38000 Grenoble, France; CEA, INAC-SCIB, Laboratoire Lésions des Acides Nucléiques, 17 rue des Martyrs, F-38054 Grenoble, France
| | - Mathilde Biola-Clier
- Université Grenoble-Alpes, INAC-LCIB, Laboratoire Lésions des Acides Nucléiques, 17 rue des Martyrs, F-38000 Grenoble, France; CEA, INAC-SCIB, Laboratoire Lésions des Acides Nucléiques, 17 rue des Martyrs, F-38054 Grenoble, France
| | - Laure Bobyk
- Université Grenoble-Alpes, INAC-LCIB, Laboratoire Lésions des Acides Nucléiques, 17 rue des Martyrs, F-38000 Grenoble, France; CEA, INAC-SCIB, Laboratoire Lésions des Acides Nucléiques, 17 rue des Martyrs, F-38054 Grenoble, France
| | - Véronique Collin-Faure
- CEA Grenoble, iRTSV/CBM, Laboratory of Chemistry and Biology of Metals, Grenoble, France
| | - Hélène Diemer
- Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), Université de Strasbourg, IPHC, 25 rue Becquerel 67087 Strasbourg, France; CNRS, UMR7178, 67037 Strasbourg, France
| | - Jean-Marc Strub
- Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), Université de Strasbourg, IPHC, 25 rue Becquerel 67087 Strasbourg, France; CNRS, UMR7178, 67037 Strasbourg, France
| | - Sarah Cianferani
- Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), Université de Strasbourg, IPHC, 25 rue Becquerel 67087 Strasbourg, France; CNRS, UMR7178, 67037 Strasbourg, France
| | - Alain Van Dorsselaer
- Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), Université de Strasbourg, IPHC, 25 rue Becquerel 67087 Strasbourg, France; CNRS, UMR7178, 67037 Strasbourg, France
| | | | - Thierry Rabilloud
- CNRS UMR 5249, Laboratory of Chemistry and Biology of Metals, Grenoble, France.
| | - Marie Carriere
- Université Grenoble-Alpes, INAC-LCIB, Laboratoire Lésions des Acides Nucléiques, 17 rue des Martyrs, F-38000 Grenoble, France; CEA, INAC-SCIB, Laboratoire Lésions des Acides Nucléiques, 17 rue des Martyrs, F-38054 Grenoble, France.
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16
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Luche S, Eymard-Vernain E, Diemer H, Van Dorsselaer A, Rabilloud T, Lelong C. Zinc oxide induces the stringent response and major reorientations in the central metabolism of Bacillus subtilis. J Proteomics 2015. [PMID: 26211718 DOI: 10.1016/j.jprot.2015.07.018] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Microorganisms, such as bacteria, are one of the first targets of nanoparticles in the environment. In this study, we tested the effect of two nanoparticles, ZnO and TiO2, with the salt ZnSO4 as the control, on the Gram-positive bacterium Bacillus subtilis by 2D gel electrophoresis-based proteomics. Despite a significant effect on viability (LD50), TiO2 NPs had no detectable effect on the proteomic pattern, while ZnO NPs and ZnSO4 significantly modified B. subtilis metabolism. These results allowed us to conclude that the effects of ZnO observed in this work were mainly attributable to Zn dissolution in the culture media. Proteomic analysis highlighted twelve modulated proteins related to central metabolism: MetE and MccB (cysteine metabolism), OdhA, AspB, IolD, AnsB, PdhB and YtsJ (Krebs cycle) and XylA, YqjI, Drm and Tal (pentose phosphate pathway). Biochemical assays, such as free sulfhydryl, CoA-SH and malate dehydrogenase assays corroborated the observed central metabolism reorientation and showed that Zn stress induced oxidative stress, probably as a consequence of thiol chelation stress by Zn ions. The other patterns affected by ZnO and ZnSO4 were the stringent response and the general stress response. Nine proteins involved in or controlled by the stringent response showed a modified expression profile in the presence of ZnO NPs or ZnSO4: YwaC, SigH, YtxH, YtzB, TufA, RplJ, RpsB, PdhB and Mbl. An increase in the ppGpp concentration confirmed the involvement of the stringent response during a Zn stress. All these metabolic reorientations in response to Zn stress were probably the result of complex regulatory mechanisms including at least the stringent response via YwaC.
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Affiliation(s)
- Sylvie Luche
- Pro-MD team, Université Joseph Fourier, CEA Grenoble, iRTSV, Laboratoire de Chimie et Biologie des Métaux, UMR CNRS-CEA-UJF, Grenoble, France
| | - Elise Eymard-Vernain
- Pro-MD team, Université Joseph Fourier, CEA Grenoble, iRTSV, Laboratoire de Chimie et Biologie des Métaux, UMR CNRS-CEA-UJF, Grenoble, France
| | - Hélène Diemer
- Laboratoire de Spectrométrie de Masse Bio-Organique, IPHC, Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg, France; CNRS, UMR7178, 67087 Strasbourg, France
| | - Alain Van Dorsselaer
- Laboratoire de Spectrométrie de Masse Bio-Organique, IPHC, Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg, France; CNRS, UMR7178, 67087 Strasbourg, France
| | - Thierry Rabilloud
- Pro-MD team, UMR CNRS 5249, Laboratoire de Chimie et Biologie des Métaux, UMR CNRS-CEA-UJF, Grenoble, France
| | - Cécile Lelong
- Pro-MD team, Université Joseph Fourier, CEA Grenoble, iRTSV, Laboratoire de Chimie et Biologie des Métaux, UMR CNRS-CEA-UJF, Grenoble, France.
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17
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Triboulet S, Aude-Garcia C, Armand L, Collin-Faure V, Chevallet M, Diemer H, Gerdil A, Proamer F, Strub JM, Habert A, Herlin N, Van Dorsselaer A, Carrière M, Rabilloud T. Comparative proteomic analysis of the molecular responses of mouse macrophages to titanium dioxide and copper oxide nanoparticles unravels some toxic mechanisms for copper oxide nanoparticles in macrophages. PLoS One 2015; 10:e0124496. [PMID: 25902355 PMCID: PMC4406518 DOI: 10.1371/journal.pone.0124496] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2014] [Accepted: 03/03/2015] [Indexed: 12/12/2022] Open
Abstract
Titanium dioxide and copper oxide nanoparticles are more and more widely used because of their catalytic properties, of their light absorbing properties (titanium dioxide) or of their biocidal properties (copper oxide), increasing the risk of adverse health effects. In this frame, the responses of mouse macrophages were studied. Both proteomic and targeted analyses were performed to investigate several parameters, such as phagocytic capacity, cytokine release, copper release, and response at sub toxic doses. Besides titanium dioxide and copper oxide nanoparticles, copper ions were used as controls. We also showed that the overall copper release in the cell does not explain per se the toxicity observed with copper oxide nanoparticles. In addition, both copper ion and copper oxide nanoparticles, but not titanium oxide, induced DNA strands breaks in macrophages. As to functional responses, the phagocytic capacity was not hampered by any of the treatments at non-toxic doses, while copper ion decreased the lipopolysaccharide-induced cytokine and nitric oxide productions. The proteomic analyses highlighted very few changes induced by titanium dioxide nanoparticles, but an induction of heme oxygenase, an increase of glutathione synthesis and a decrease of tetrahydrobiopterin in response to copper oxide nanoparticles. Subsequent targeted analyses demonstrated that the increase in glutathione biosynthesis and the induction of heme oxygenase (e.g. by lovastatin/monacolin K) are critical for macrophages to survive a copper challenge, and that the intermediates of the catecholamine pathway induce a strong cross toxicity with copper oxide nanoparticles and copper ions.
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Affiliation(s)
- Sarah Triboulet
- Laboratory of Chemistry and Biology of Metals, Univ. Grenoble Alpes, Grenoble, France
| | - Catherine Aude-Garcia
- Laboratory of Chemistry and Biology of Metals, CEA/ iRTSV, Grenoble, France
- Laboratory of Chemistry and Biology of Metals, CNRS UMR5249, Grenoble, France
| | - Lucie Armand
- Service de Chimie Inorganique et Biologique, Univ. Grenoble Alpes & CEA, Grenoble, France
| | | | - Mireille Chevallet
- Laboratory of Chemistry and Biology of Metals, CEA/ iRTSV, Grenoble, France
| | - Hélène Diemer
- Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), Université de Strasbourg & CNRS UMR 7178, Institut Pluridisciplinaire Hubert Curien, Strasbourg, France
| | - Adèle Gerdil
- Laboratoire Francis Perrin (Unité de recherche Associée 2453), Commissariat à l’Energie Atomique, CEA-Saclay 91191 Gif/Yvette, France
| | - Fabienne Proamer
- Etablissement Français du Sang-Alsace, Unité MIxte de recherche S949 Institut National de la Santé Et de la Recherche Médicale (INSERM)-Université de Strasbourg, Strasbourg, France
| | - Jean-Marc Strub
- Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), Université de Strasbourg & CNRS UMR 7178, Institut Pluridisciplinaire Hubert Curien, Strasbourg, France
| | - Aurélie Habert
- Laboratoire Francis Perrin (Unité de recherche Associée 2453), Commissariat à l’Energie Atomique, CEA-Saclay 91191 Gif/Yvette, France
| | - Nathalie Herlin
- Laboratoire Francis Perrin (Unité de recherche Associée 2453), Commissariat à l’Energie Atomique, CEA-Saclay 91191 Gif/Yvette, France
| | - Alain Van Dorsselaer
- Laboratoire de Spectrométrie de Masse BioOrganique (LSMBO), Université de Strasbourg & CNRS UMR 7178, Institut Pluridisciplinaire Hubert Curien, Strasbourg, France
| | - Marie Carrière
- Service de Chimie Inorganique et Biologique, Univ. Grenoble Alpes & CEA, Grenoble, France
| | - Thierry Rabilloud
- Laboratory of Chemistry and Biology of Metals, CNRS UMR5249, Grenoble, France
- * E-mail:
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18
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Scaife C, McManus CA, Donoghue PM, Dunn MJ. Co-detection of Target and Total Protein by CyDye Labeling and Fluorescent ECL Plex Immunoblotting in a Standard Proteomics Workflow. Methods Mol Biol 2015; 1314:139-149. [PMID: 26139262 DOI: 10.1007/978-1-4939-2718-0_15] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/04/2023]
Abstract
The qualitative and quantitative capabilities of 2-D electrophoresis and its use in widespread proteome analysis have been revolutionized over the past decade with the introduction of differential gel electrophoresis commonly known as DIGE. This highly sensitive CyDye protein labeling technique now attempts to advance conventional western blotting by the combination of DIGE labeling with ECL Plex CyDye conjugated secondary antibodies. The ability of this method to simultaneously visualize the total protein expression profile as well as the specific immunodetection of an individual protein species will significantly aid protein validation following 2-D gel separation by confirming the exact location of proteins of interest. This simple, rapid, and reproducible technique is demonstrated by 1-D and 2-D electrophoresis through the detection of the small 27 kDa heat shock protein (hsp 27), a protein known to be expressed in the human heart, from a complex cardiac protein extract.
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Affiliation(s)
- Caitriona Scaife
- Proteome Research Centre, UCD Conway Institute of Biomolecular and Biomedical Research, Dublin 4, Ireland
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19
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Santucci L, Bruschi M, Ghiggeri GM, Candiano G. The latest advancements in proteomic two-dimensional gel electrophoresis analysis applied to biological samples. Methods Mol Biol 2015; 1243:103-125. [PMID: 25384742 DOI: 10.1007/978-1-4939-1872-0_6] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/04/2023]
Abstract
Two-dimensional gel electrophoresis (2DE) is one of the fundamental approaches in proteomics for the separation and visualization of complex protein mixtures. Proteins can be analyzed by 2DE using isoelectric focusing (IEF) in the first dimension, combined to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension, gel staining (silver and Coomassie), image analysis, and 2DE gel database. High-resolution 2DE can resolve up to 5,000 different proteins simultaneously (∼2,000 proteins routinely), and detect and quantify <1 ng of protein per spot. Here, we describe the latest developments for a more complete analysis of biological fluids.
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Affiliation(s)
- Laura Santucci
- Laboratory on Pathophysiology of Uremia, Istituto Giannina Gaslini, Largo G. Gaslini 5, Genoa, Italy
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20
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Abstract
Sample preparation is one of the key technologies for successful two-dimensional electrophoresis (2DE). Due to the great diversity of protein sample types and sources, no single sample preparation method works with all proteins; for any sample the optimum procedure must be determined empirically. This review is meant to provide a broad overview of the most important principles in sample preparation in order to avoid a multitude of possible pitfalls. Sample preparation protocols from the expert in the field were screened and evaluated. On the basis of these protocols and my own comprehensive practical experience important guidelines are given in this review. The presented guidelines will facilitate straightforward protocol development for researchers new to gel-based proteomics. In addition the available choices are rationalized in order to successfully prepare a protein sample for 2DE separations. The strategies described here are not limited to 2DE and can also be applied to other protein separation techniques.
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Affiliation(s)
- Anton Posch
- Bio-Rad Laboratories GmbH , Heidemannstrasse Munich , Germany
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21
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Triboulet S, Aude-Garcia C, Armand L, Gerdil A, Diemer H, Proamer F, Collin-Faure V, Habert A, Strub JM, Hanau D, Herlin N, Carrière M, Van Dorsselaer A, Rabilloud T. Analysis of cellular responses of macrophages to zinc ions and zinc oxide nanoparticles: a combined targeted and proteomic approach. NANOSCALE 2014; 6:6102-6114. [PMID: 24788578 DOI: 10.1039/c4nr00319e] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/03/2023]
Abstract
Two different zinc oxide nanoparticles, as well as zinc ions, are used to study the cellular responses of the RAW 264 macrophage cell line. A proteomic screen is used to provide a wide view of the molecular effects of zinc, and the most prominent results are cross-validated by targeted studies. Furthermore, the alteration of important macrophage functions (e.g. phagocytosis) by zinc is also investigated. The intracellular dissolution/uptake of zinc is also studied to further characterize zinc toxicity. Zinc oxide nanoparticles dissolve readily in the cells, leading to high intracellular zinc concentrations, mostly as protein-bound zinc. The proteomic screen reveals a rather weak response in the oxidative stress response pathway, but a strong response both in the central metabolism and in the proteasomal protein degradation pathway. Targeted experiments confirm that carbohydrate catabolism and proteasome are critical determinants of sensitivity to zinc, which also induces DNA damage. Conversely, glutathione levels and phagocytosis appear unaffected at moderately toxic zinc concentrations.
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Affiliation(s)
- Sarah Triboulet
- Univ. Grenoble Alpes, Laboratory of Chemistry and Biology of Metals, UMR CNRS-CEA-UJF 5249, iRTSV/LCBM, CEA Grenoble, 17 rue des martyrs, F-38054 Grenoble Cedex 9, France.
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22
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Nebija D, Noe CR, Urban E, Lachmann B. Quality control and stability studies with the monoclonal antibody, trastuzumab: application of 1D- vs. 2D-gel electrophoresis. Int J Mol Sci 2014; 15:6399-411. [PMID: 24739811 PMCID: PMC4013636 DOI: 10.3390/ijms15046399] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2013] [Revised: 03/10/2014] [Accepted: 03/31/2014] [Indexed: 12/05/2022] Open
Abstract
Recombinant monoclonal antibodies (rmAbs) are medicinal products obtained by rDNA technology. Consequently, like other biopharmaceuticals, they require the extensive and rigorous characterization of the quality attributes, such as identity, structural integrity, purity and stability. The aim of this work was to study the suitability of gel electrophoresis for the assessment of charge heterogeneity, post-translational modifications and the stability of the therapeutic, recombinant monoclonal antibody, trastuzumab. One-dimensional, SDS-PAGE, under reducing and non-reducing conditions, and two-dimensional gel electrophoresis were used for the determination of molecular mass (Mr), the isoelectric point (pI), charge-related isoform patterns and the stability of trastuzumab, subjected to stressed degradation and long-term conditions. For the assessment of the influence of glycosylation in the charge heterogeneity pattern of trastuzumab, an enzymatic deglycosylation study has been performed using N-glycosidase F and sialidase, whereas carboxypeptidase B was used for the lysine truncation study. Experimental data documented that 1D and 2D gel electrophoresis represent fast and easy methods to evaluate the quality of biological medicinal products. Important stability parameters, such as the protein aggregation, can be assessed, as well.
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Affiliation(s)
- Dashnor Nebija
- Department of Pharmaceutical Chemistry, Medical Faculty, Rr. Bulevardi i Deshmoreve, n.n. 10000 Pristina, Kosovo.
| | - Christian R Noe
- Department of Pharmaceutical Chemistry, Faculty of Life Sciences, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria.
| | - Ernst Urban
- Department of Pharmaceutical Chemistry, Faculty of Life Sciences, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria.
| | - Bodo Lachmann
- Department of Pharmaceutical Chemistry, Faculty of Life Sciences, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria.
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23
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Sabater-Jara AB, Almagro L, Belchí-Navarro S, Martínez-Esteso MJ, Youssef SM, Casado-Vela J, Vera-Urbina JC, Sellés-Marchart S, Bru-Martínez R, Pedreño MA. Suspension-cultured plant cells as a tool to analyze the extracellular proteome. Methods Mol Biol 2013; 1072:407-33. [PMID: 24136538 DOI: 10.1007/978-1-62703-631-3_29] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/20/2023]
Abstract
Suspension-cultured cells (SCC) are generally considered the most suitable cell systems to carry out scientific studies, including the extracellular proteome (secretome). SCC are initiated by transferring friable callus fragments into flasks containing liquid culture medium for cell biomass growth, and they are maintained in an orbital shaker to supply the sufficient oxygen that allows cell growth. SCC increase rapidly during the exponential phase and after 10-20 days (depending on the cell culture nature), the growth rate starts to decrease due to limitation of nutrients, and to maintain for decades these kinds of cell cultures is needed to transfer a portion of these SCC into a fresh culture medium. Despite the central role played by extracellular proteins in most processes that control growth and development, the secretome has been less well characterized than other subcellular compartments, meaning that our understanding of the cell wall physiology is still very limited. Useful proteomic tools have emerged in recent years to unravel metabolic network that occurs in cell walls. With the recent progress made in mass spectrometry technology, it has become feasible to identify proteins from a given organ, tissue, cells, or even a subcellular compartment. Compared with other methods used to isolate cell wall proteins, the spent medium of SCC provides a convenient, continuous, and reliable and unique source of extracellular proteins. Therefore, this biological system could be used as a large-scale cell culture from which these proteins can be secreted, easily separated from cells without cell disruption, and so, without any cytosolic contamination, easily recovered from the extracellular medium. This nondestructive cell wall proteome approach discloses a set of proteins that are specifically expressed in the remodelling of the cell wall architecture and stress defense.
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Affiliation(s)
- Ana B Sabater-Jara
- Plant Peroxidases Group, Department of Plant Physiology, Faculty of Biology, University of Murcia, Murcia, Spain
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24
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Triboulet S, Aude-Garcia C, Carrière M, Diemer H, Proamer F, Habert A, Chevallet M, Collin-Faure V, Strub JM, Hanau D, Van Dorsselaer A, Herlin-Boime N, Rabilloud T. Molecular responses of mouse macrophages to copper and copper oxide nanoparticles inferred from proteomic analyses. Mol Cell Proteomics 2013; 12:3108-22. [PMID: 23882024 DOI: 10.1074/mcp.m113.030742] [Citation(s) in RCA: 55] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
The molecular responses of macrophages to copper-based nanoparticles have been investigated via a combination of proteomic and biochemical approaches, using the RAW264.7 cell line as a model. Both metallic copper and copper oxide nanoparticles have been tested, with copper ion and zirconium oxide nanoparticles used as controls. Proteomic analysis highlighted changes in proteins implicated in oxidative stress responses (superoxide dismutases and peroxiredoxins), glutathione biosynthesis, the actomyosin cytoskeleton, and mitochondrial proteins (especially oxidative phosphorylation complex subunits). Validation studies employing functional analyses showed that the increases in glutathione biosynthesis and in mitochondrial complexes observed in the proteomic screen were critical to cell survival upon stress with copper-based nanoparticles; pharmacological inhibition of these two pathways enhanced cell vulnerability to copper-based nanoparticles, but not to copper ions. Furthermore, functional analyses using primary macrophages derived from bone marrow showed a decrease in reduced glutathione levels, a decrease in the mitochondrial transmembrane potential, and inhibition of phagocytosis and of lipopolysaccharide-induced nitric oxide production. However, only a fraction of these effects could be obtained with copper ions. In conclusion, this study showed that macrophage functions are significantly altered by copper-based nanoparticles. Also highlighted are the cellular pathways modulated by cells for survival and the exemplified cross-toxicities that can occur between copper-based nanoparticles and pharmacological agents.
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Affiliation(s)
- Sarah Triboulet
- Pro-MD team, Laboratoire de Chimie et Biologie des Métaux, UMR CNRS-CEA-UJF, Université Joseph Fourier, Grenoble 38054, France
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Hadji Sfaxi I, Ezzine A, Coquet L, Cosette P, Jouenne T, Marzouki MN. Combined proteomic and molecular approaches for cloning and characterization of copper-zinc superoxide dismutase (Cu, Zn-SOD2) from garlic (Allium sativum). Mol Biotechnol 2013; 52:49-58. [PMID: 22161312 DOI: 10.1007/s12033-011-9473-8] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/14/2022]
Abstract
Superoxide dismutases (SODs; EC 1.15.1.1) are key enzymes in the cells protection against oxidant agents. Thus, SODs play a major role in the protection of aerobic organisms against oxygen-mediated damages. Three SOD isoforms were previously identified by zymogram staining from Allium sativum bulbs. The purified Cu, Zn-SOD2 shows an antagonist effect to an anticancer drug and alleviate cytotoxicity inside tumor cells lines B16F0 (mouse melanoma cells) and PAE (porcine aortic endothelial cells). To extend the characterization of Allium SODs and their corresponding genes, a proteomic approach was applied involving two-dimensional gel electrophoresis and LC-MS/MS analyses. From peptide sequence data obtained by mass spectrometry and sequences homologies, primers were defined and a cDNA fragment of 456 bp was amplified by RT-PCR. The cDNA nucleotide sequence analysis revealed an open reading frame coding for 152 residues. The deduced amino acid sequence showed high identity (82-87%) with sequences of Cu, Zn-SODs from other plant species. Molecular analysis was achieved by a protein 3D structural model.
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Affiliation(s)
- Imen Hadji Sfaxi
- Department of Bioengineering, National Institute of Applied Sciences and Technology, University of Carthage, 676-1080 Tunis Cedex, Tunisia.
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Lelong C, Chevallet M, Diemer H, Luche S, Van Dorsselaer A, Rabilloud T. Improved proteomic analysis of nuclear proteins, as exemplified by the comparison of two myeloid cell lines nuclear proteomes. J Proteomics 2012; 77:577-602. [DOI: 10.1016/j.jprot.2012.09.034] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2012] [Revised: 09/18/2012] [Accepted: 09/22/2012] [Indexed: 02/04/2023]
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Khojasteh SC, Hartley DP, Ford KA, Uppal H, Oishi S, Nelson SD. Characterization of Rat Liver Proteins Adducted by Reactive Metabolites of Menthofuran. Chem Res Toxicol 2012; 25:2301-9. [DOI: 10.1021/tx300144d] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Affiliation(s)
- S. Cyrus Khojasteh
- Drug Metabolism
and Pharmacokinetics, Genentech, Inc.,
1 DNA Way MS 412a, South San Francisco,
California 94080, United States
| | | | - Kevin A. Ford
- Safety Assessment, Genentech, Inc., 1
DNA Way MS 59, South San Francisco,
California 94080, United States
| | - Hirdesh Uppal
- Safety Assessment, Genentech, Inc., 1
DNA Way MS 59, South San Francisco,
California 94080, United States
| | - Shimako Oishi
- Pharma Products Group, Abbott Japan Co., Ltd., Tokyo 108-6303, Japan
| | - Sidney D. Nelson
- Department of Medicinal Chemistry,
School of Pharmacy, University of Washington, Box 357610, Seattle, Washington 98195, United States
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Föcking M, Chen WQ, Dicker P, Dunn MJ, Lubec G, Cotter DR. Proteomic analysis of human hippocampus shows differential protein expression in the different hippocampal subfields. Proteomics 2012; 12:2477-81. [DOI: 10.1002/pmic.201200031] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Affiliation(s)
- Melanie Föcking
- Department of Psychiatry; Royal College of Surgeons in Ireland; Dublin; Ireland
| | - Wei-Qiang Chen
- Department of Pediatrics; Medical University of Vienna; Vienna; Austria
| | - Patrick Dicker
- Department of Epidemiology; Royal College of Surgeons in Ireland; Dublin; Ireland
| | - Michael J. Dunn
- Proteome Research Centre; UCD Conway Institute of Biomolecular and Biomedical Research, School of Medicine and Medical Sciences; Dublin; Ireland
| | - Gert Lubec
- Department of Pediatrics; Medical University of Vienna; Vienna; Austria
| | - David R. Cotter
- Department of Psychiatry; Royal College of Surgeons in Ireland; Dublin; Ireland
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Kuwana R, Yamamoto N. Increases in GroES and GroEL from Lactobacillus acidophilus L-92 in response to a decrease in medium pH, and changes in cytokine release from splenocytes: Transcriptome and proteome analyses. J Biosci Bioeng 2012; 114:9-16. [DOI: 10.1016/j.jbiosc.2012.02.010] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2011] [Revised: 02/06/2012] [Accepted: 02/13/2012] [Indexed: 11/24/2022]
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Keenan J, Manning G, Elia G, Dunn MJ, Orr DF, Pierscionek BK. Crystallin distribution patterns in Litoria infrafrenata and Phyllomedusa sauvagei lenses. Proteomics 2012; 12:1830-43. [PMID: 22623336 DOI: 10.1002/pmic.201100393] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The eye lens remains transparent because of soluble lens proteins known as crystallins. For years γ-crystallins have been known as the main lens proteins in lower vertebrates such as fish and amphibians. The unique growth features of the lens render it an ideal structure to study ageing; few studies have examined such changes in anuran lenses. This study aimed to investigate protein distribution patterns in Litoria infrafrenata and Phyllomedusa sauvagei species. Lenses were fractionated into concentric layers by controlled dissolution. Water-soluble proteins were separated into high (HMW), middle (MMW) and low molecular weight (LMW) fractions by size-exclusion HPLC and constituents of each protein class revealed by 1DE and 2DE. Spots were selected from 2DE gels on the basis of known ranges of subunit molecular weights and pH ranges and were identified by MALDI-TOF/TOF MS following trypsin digestion. Comparable lens distribution patterns were found for each species studied. Common crystallins were detected in both species; the most prominent of these was γ-crystallin. Towards the lens centre, there was a decrease in α- and β-crystallin proportions and an increase in γ-crystallins. Subunits representing taxon-specific crystallins demonstrating strong sequence homology with ζ-crystallin/quinone oxidoreductase were found in both L. infrafrenata and P. sauvagei lenses. Further work is needed to determine which amphibians have taxon-specific crystallins, their evolutionary origins, and their function.
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Affiliation(s)
- Jonathan Keenan
- School of Biomedical Sciences, University of Ulster, Coleraine, UK
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Abstract
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is based on the combination of two orthogonal separation techniques. In the first dimension, proteins are separated by their isoelectric point, a technique known as isoelectric focusing (IEF). There are two important variants of IEF, which are carrier-ampholine (CA)-based IEF and immobilized pH gradient (IPG)-based IEF. In the second dimension, proteins are further separated by their electrophoretic mobility using SDS-PAGE. Finally, proteins can be visualized and quantified by different staining procedures, such as Coomassie, silver, or fluorescence staining. This chapter gives detailed protocols for 2D-PAGE, using both CA- and IPG-based separation in the first dimension.
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Affiliation(s)
- Caroline May
- Department of Medical Proteomics/Bionalaytics, Medizinisches Proteom-Center, Ruhr-Universität Bochum, Bochum, Germany.
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Nebija D, Kopelent-Frank H, Urban E, Noe CR, Lachmann B. Comparison of two-dimensional gel electrophoresis patterns and MALDI-TOF MS analysis of therapeutic recombinant monoclonal antibodies trastuzumab and rituximab. J Pharm Biomed Anal 2011; 56:684-91. [DOI: 10.1016/j.jpba.2011.07.006] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2011] [Revised: 05/25/2011] [Accepted: 07/06/2011] [Indexed: 01/14/2023]
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Characterization of adhesive molecule with affinity to Caco-2 cells in Lactobacillus acidophilus by proteome analysis. J Biosci Bioeng 2011; 112:333-7. [DOI: 10.1016/j.jbiosc.2011.06.001] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2011] [Revised: 05/23/2011] [Accepted: 06/07/2011] [Indexed: 01/26/2023]
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DIGE analysis of proteome changes accompanying large resveratrol production by grapevine (Vitis vinifera cv. Gamay) cell cultures in response to methyl-β-cyclodextrin and methyl jasmonate elicitors. J Proteomics 2011; 74:1421-36. [DOI: 10.1016/j.jprot.2011.02.035] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2010] [Revised: 02/24/2011] [Accepted: 02/28/2011] [Indexed: 01/17/2023]
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Yasui Y, Tanaka T. Protein expression analysis of inflammation-related colon carcinogenesis. J Carcinog 2011; 8:10. [PMID: 19491504 PMCID: PMC2699605 DOI: 10.4103/1477-3163.51851] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Background: Chronic inflammation is a risk factor for colorectal cancer (CRC) development. The aim of this study was to determine the differences in protein expression between CRC and the surrounding nontumorous colonic tissues in the mice that received azoxymethane (AOM) and dextran sodium sulfate (DSS) using a proteomic analysis. Materials and Methods: Male ICR mice were given a single intraperitoneal injection of AOM (10 mg/kg body weight), followed by 2% (w/v) DSS in their drinking water for seven days, starting one week after the AOM injection. Colonic adenocarcinoma developed after 20 weeks and a proteomics analysis based on two-dimensional gel electrophoresis and ultraflex TOF/TOF mass spectrometry was conducted in the cancerous and nontumorous tissue specimens. Results: The proteomic analysis revealed 21 differentially expressed proteins in the cancerous tissues in comparison to the nontumorous tissues. There were five markedly increased proteins (beta-tropomyosin, tropomyosin 1 alpha isoform b, S100 calcium binding protein A9, and an unknown protein) and 16 markedly decreased proteins (Car1 proteins, selenium-binding protein 1, HMG-CoA synthase, thioredoxin 1, 1 Cys peroxiredoxin protein 2, Fcgbp protein, Cytochrome c oxidase, subunit Va, ETHE1 protein, and 7 unknown proteins). Conclusions: There were 21 differentially expressed proteins in the cancerous tissues of the mice that received AOM and DSS. Their functions include metabolism, the antioxidant system, oxidative stress, mucin production, and inflammation. These findings may provide new insights into the mechanisms of inflammation-related colon carcinogenesis and the establishment of novel therapies and preventative strategies to treat carcinogenesis in the inflamed colon.
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Affiliation(s)
- Yumiko Yasui
- Department of Oncologic Pathology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan.
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Chevallet M, Aude-Garcia C, Lelong C, Candéias S, Luche S, Collin-Faure V, Triboulet S, Diallo D, Diemer H, Dorsselaer AV, Rabilloud T. Effects of nanoparticles on murine macrophages. ACTA ACUST UNITED AC 2011. [DOI: 10.1088/1742-6596/304/1/012034] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
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Two-dimensional gel electrophoresis in proteomics: a tutorial. J Proteomics 2011; 74:1829-41. [PMID: 21669304 DOI: 10.1016/j.jprot.2011.05.040] [Citation(s) in RCA: 170] [Impact Index Per Article: 12.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2011] [Revised: 05/23/2011] [Accepted: 05/26/2011] [Indexed: 12/12/2022]
Abstract
Two-dimensional electrophoresis of proteins has preceded, and accompanied, the birth of proteomics. Although it is no longer the only experimental scheme used in modern proteomics, it still has distinct features and advantages. The purpose of this tutorial paper is to guide the reader through the history of the field, then through the main steps of the process, from sample preparation to in-gel detection of proteins, commenting the constraints and caveats of the technique. Then the limitations and positive features of two-dimensional electrophoresis are discussed (e.g. its unique ability to separate complete proteins and its easy interfacing with immunoblotting techniques), so that the optimal type of applications of this technique in current and future proteomics can be perceived. This is illustrated by a detailed example taken from the literature and commented in detail. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 2).
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38
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Nebija D, Urban E, Stessl M, Noe CR, Lachmann B. 2-DE and MALDI-TOF-MS analysis of therapeutic fusion protein abatacept. Electrophoresis 2011; 32:1438-43. [DOI: 10.1002/elps.201100029] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2011] [Revised: 02/22/2011] [Accepted: 03/04/2011] [Indexed: 11/08/2022]
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40
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Schieb H, Spitzer P, Riess V, Wiltfang J, Klafki HW. A method to prevent cross contamination during 2-DE by β-amyloid peptides. Proteomics 2011; 10:3539-43. [PMID: 20827735 DOI: 10.1002/pmic.201000227] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
A method for the efficient decontamination of aluminium oxide ceramic 2-DE focusing trays from β-amyloid peptides (Aβ) is reported. As these contaminations were resistant to the standard cleaning procedures, additional harsh cleaning steps were necessary for their efficient removal. Our observations suggest that specific surface properties affect the degree of adsorption of the Aβ-peptides. "Surface catalysed amyloid aggregation" in the aluminium oxide ceramic trays is proposed as a possible underlying mechanism for the occurrence of proteinase K-resistant forms of Aβ.
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Affiliation(s)
- Heinke Schieb
- Department of Psychiatry and Psychotherapy, University of Duisburg-Essen, LVR-Klinikum, Essen, Germany
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41
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Van Steendam K, Tilleman K, De Ceuleneer M, De Keyser F, Elewaut D, Deforce D. Citrullinated vimentin as an important antigen in immune complexes from synovial fluid of rheumatoid arthritis patients with antibodies against citrullinated proteins. Arthritis Res Ther 2010; 12:R132. [PMID: 20609218 PMCID: PMC2945022 DOI: 10.1186/ar3070] [Citation(s) in RCA: 77] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2009] [Revised: 06/10/2010] [Accepted: 07/07/2010] [Indexed: 11/10/2022] Open
Abstract
INTRODUCTION Rheumatoid arthritis (RA) is an inflammatory disease, which results in destruction of the joint. The presence of immune complexes (IC) in serum and synovial fluid of RA patients might contribute to this articular damage through different mechanisms, such as complement activation. Therefore, identification of the antigens from these IC is important to gain more insight into the pathogenesis of RA. Since RA patients have antibodies against citrullinated proteins (ACPA) in their serum and synovial fluid (SF) and since elevated levels of citrullinated proteins are detected in the joints of RA patients, citrullinated antigens are possibly present in IC from RA patients. METHODS IC from serum of healthy persons, serum of RA patients and IC from synovial fluid of RA patients and Spondyloarthropathy (SpA) patients were isolated by immunoprecipitation. Identification of the antigens was performed by SDS-PAGE, mass spectrometry and immunodetection. The presence of citrullinated proteins was evaluated by anti-modified citrulline (AMC) staining. RESULTS Circulating IC in the serum of RA patients and healthy controls contain fibrinogenβ and fibronectin, both in a non-citrullinated form. Additionally, in IC isolated from RA SF, fibrinogenγ and vimentin were identified as well. More importantly, vimentin and a minor portion of fibrinogenβ were found to be citrullinated in the isolated complexes. Moreover these citrullinated antigens were only found in ACPA+ patients. No citrullinated antigens were found in IC from SF of SpA patients. CONCLUSIONS Citrullinated fibrinogenβ and citrullinated vimentin were found in IC from SF of ACPA+ RA patients, while no citrullinated antigens were found in IC from SF of ACPA- RA patients or SpA patients or in IC from serum of RA patients or healthy volunteers. The identification of citrullinated vimentin as a prominent citrullinated antigen in IC from SF of ACPA+ RA patients strengthens the hypothesis that citrullinated vimentin plays an important role in the pathogenesis of RA.
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Two-dimensional gel electrophoresis in proteomics: Past, present and future. J Proteomics 2010; 73:2064-77. [PMID: 20685252 DOI: 10.1016/j.jprot.2010.05.016] [Citation(s) in RCA: 292] [Impact Index Per Article: 19.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2010] [Revised: 05/20/2010] [Accepted: 05/25/2010] [Indexed: 12/14/2022]
Abstract
Two-dimensional gel electrophoresis has been instrumental in the birth and developments of proteomics, although it is no longer the exclusive separation tool used in the field of proteomics. In this review, a historical perspective is made, starting from the days where two-dimensional gels were used and the word proteomics did not even exist. The events that have led to the birth of proteomics are also recalled, ending with a description of the now well-known limitations of two-dimensional gels in proteomics. However, the often-underestimated advantages of two-dimensional gels are also underlined, leading to a description of how and when to use two-dimensional gels for the best in a proteomics approach. Taking support of these advantages (robustness, resolution, and ability to separate entire, intact proteins), possible future applications of this technique in proteomics are also mentioned.
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Furuhashi T, Miksik I, Smrz M, Germann B, Nebija D, Lachmann B, Noe C. Comparison of aragonitic molluscan shell proteins. Comp Biochem Physiol B Biochem Mol Biol 2010; 155:195-200. [PMID: 19932190 DOI: 10.1016/j.cbpb.2009.11.007] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2009] [Revised: 11/11/2009] [Accepted: 11/12/2009] [Indexed: 10/20/2022]
Abstract
Acidic macromolecules, as a nucleation factor for mollusc shell formation, are a major focus of research. It remains unclear, however, whether acidic macromolecules are present only in calcified shell organic matrices, and which acidic macromolecules are crucial for the nucleation process by binding to chitin as structural components. To clarify these questions, we applied 2D gel electrophoresis and amino acid analysis to soluble shell organic matrices from nacre shell, non-nacre aragonitic shell and non-calcified squid shells. The 2D gel electrophoresis results showed that the acidity of soluble proteins differs even between nacre shells, and some nacre (Haliotis gigantea) showed a basic protein migration pattern. Non-calcified shells also contained some moderately acidic proteins. The results did not support the correlation between the acidity of soluble shell proteins and shell structure.
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Affiliation(s)
- Takeshi Furuhashi
- Department of Molecular System Biology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria.
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44
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Hodgkinson VC, Eagle GL, Drew PJ, Lind MJ, Cawkwell L. Biomarkers of chemotherapy resistance in breast cancer identified by proteomics: current status. Cancer Lett 2010; 294:13-24. [PMID: 20176436 DOI: 10.1016/j.canlet.2010.01.036] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2009] [Revised: 01/26/2010] [Accepted: 01/29/2010] [Indexed: 11/26/2022]
Abstract
This review describes and discusses the advantages and limitations of proteomic approaches in the identification of biomarkers associated with chemotherapy resistance. Both gel-based (two-dimensional polyacrylamide gel electrophoresis) and gel-free (shotgun and quantitative) mass spectrometry approaches are discussed. Non-mass spectrometry approaches including antibody microarray platforms are described as complementary proteomic strategies. Methods for technical confirmation and clinical validation of putative biomarkers are presented. Use of this proteomic toolbox in the quest for biomarkers of chemotherapy resistance in breast cancer is reviewed. Technical aspects of sample selection, acquisition, storage and analysis are discussed and putative biomarkers identified through proteomic approaches are presented.
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Affiliation(s)
- Victoria C Hodgkinson
- Cancer Biology Proteomics Group, Postgraduate Medical Institute of the University of Hull, Hull, UK
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45
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Grassl J, Westbrook JA, Robinson A, Borén M, Dunn MJ, Clyne RK. Preserving the yeast proteome from sample degradation. Proteomics 2010; 9:4616-26. [PMID: 19824011 DOI: 10.1002/pmic.200800945] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Sample degradation is a common problem in all types of proteomic analyses as it generates protein and peptide fragments that can interfere with analytical results. An important step in preventing such artefacts is to preserve the native, intact proteome as early as possible during sample preparation prior to proteomic analysis. Using the budding yeast Saccharomyces cerevisiae, we have evaluated the effects of trichloroacetic acid (TCA) and thermal treatments prior to protein extraction as a means to minimise proteolysis. TCA precipitation is commonly used to inactivate proteases; thermal stabilisation is used to heat samples to approximately 95 degrees C to inactivate enzyme activity. The efficacy of these methods was also compared with that of protease inhibitors and lyophilisation. Sample integrity was assessed by 2-D PAGE and a selection of spots was identified by MS/MS. The analysis showed that TCA or thermal treatment significantly reduced the degree of degradation and that these pre-treatment protocols were more effective than treatment with either protease inhibitors or lyophilisation. This study establishes standardised sample preparation methods for the reproducible analysis of protein patterns by 2-D PAGE in yeast, and may also be applicable to other proteomic analyses such as gel-free-based quantitation methods.
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Affiliation(s)
- Julia Grassl
- UCD Conway Institute of Biomolecular and Biomedical Research, School of Biomolecular and Biomedical Science, University College Dublin, Ireland
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Wang Y, Carolan JC, Hao F, Nicholson JK, Wilkinson TL, Douglas AE. Integrated Metabonomic−Proteomic Analysis of an Insect−Bacterial Symbiotic System. J Proteome Res 2010; 9:1257-67. [DOI: 10.1021/pr9007392] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Affiliation(s)
- Yulan Wang
- State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Centre for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071, PR China, UCD School of Biology and Environmental Science, University College Dublin, Dublin, Ireland, Department of Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, South Kensington, London, SW7, 2AZ, U.K., Department of
| | - James C. Carolan
- State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Centre for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071, PR China, UCD School of Biology and Environmental Science, University College Dublin, Dublin, Ireland, Department of Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, South Kensington, London, SW7, 2AZ, U.K., Department of
| | - FuHua Hao
- State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Centre for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071, PR China, UCD School of Biology and Environmental Science, University College Dublin, Dublin, Ireland, Department of Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, South Kensington, London, SW7, 2AZ, U.K., Department of
| | - Jeremy K. Nicholson
- State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Centre for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071, PR China, UCD School of Biology and Environmental Science, University College Dublin, Dublin, Ireland, Department of Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, South Kensington, London, SW7, 2AZ, U.K., Department of
| | - Thomas L. Wilkinson
- State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Centre for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071, PR China, UCD School of Biology and Environmental Science, University College Dublin, Dublin, Ireland, Department of Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, South Kensington, London, SW7, 2AZ, U.K., Department of
| | - Angela E. Douglas
- State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Centre for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, 430071, PR China, UCD School of Biology and Environmental Science, University College Dublin, Dublin, Ireland, Department of Biomolecular Medicine, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, South Kensington, London, SW7, 2AZ, U.K., Department of
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47
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Martinez-Esteso M, Sellés-Marchart S, Vera-Urbina J, Pedreño M, Bru-Martinez R. Changes of defense proteins in the extracellular proteome of grapevine (Vitis vinifera cv. Gamay) cell cultures in response to elicitors. J Proteomics 2009; 73:331-41. [DOI: 10.1016/j.jprot.2009.10.001] [Citation(s) in RCA: 69] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2009] [Revised: 09/23/2009] [Accepted: 10/01/2009] [Indexed: 10/20/2022]
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Yamazaki H, Kuribayashi S, Inoue T, Tateno C, Nishikura Y, Oofusa K, Harada D, Naito S, Horie T, Ohta S. Approach for in Vivo Protein Binding of 5-n-Butyl-pyrazolo[1,5-a]pyrimidine Bioactivated in Chimeric Mice with Humanized Liver by Two-Dimensional Electrophoresis with Accelerator Mass Spectrometry. Chem Res Toxicol 2009; 23:152-8. [DOI: 10.1021/tx900323a] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Affiliation(s)
- Hiroshi Yamazaki
- Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan, Preclinical Assessment Department, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima 772-8601, Japan, PhoenixBio, Co., Higashi-Hiroshima, Hiroshima 739-0046, Japan, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Hiroshima 734-8553, Japan, and Towa Environment Science Co., Suminoe-ku, Osaka 559-0034, Japan
| | - Shunji Kuribayashi
- Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan, Preclinical Assessment Department, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima 772-8601, Japan, PhoenixBio, Co., Higashi-Hiroshima, Hiroshima 739-0046, Japan, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Hiroshima 734-8553, Japan, and Towa Environment Science Co., Suminoe-ku, Osaka 559-0034, Japan
| | - Tae Inoue
- Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan, Preclinical Assessment Department, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima 772-8601, Japan, PhoenixBio, Co., Higashi-Hiroshima, Hiroshima 739-0046, Japan, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Hiroshima 734-8553, Japan, and Towa Environment Science Co., Suminoe-ku, Osaka 559-0034, Japan
| | - Chise Tateno
- Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan, Preclinical Assessment Department, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima 772-8601, Japan, PhoenixBio, Co., Higashi-Hiroshima, Hiroshima 739-0046, Japan, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Hiroshima 734-8553, Japan, and Towa Environment Science Co., Suminoe-ku, Osaka 559-0034, Japan
| | - Yasufumi Nishikura
- Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan, Preclinical Assessment Department, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima 772-8601, Japan, PhoenixBio, Co., Higashi-Hiroshima, Hiroshima 739-0046, Japan, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Hiroshima 734-8553, Japan, and Towa Environment Science Co., Suminoe-ku, Osaka 559-0034, Japan
| | - Ken Oofusa
- Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan, Preclinical Assessment Department, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima 772-8601, Japan, PhoenixBio, Co., Higashi-Hiroshima, Hiroshima 739-0046, Japan, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Hiroshima 734-8553, Japan, and Towa Environment Science Co., Suminoe-ku, Osaka 559-0034, Japan
| | - Daisuke Harada
- Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan, Preclinical Assessment Department, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima 772-8601, Japan, PhoenixBio, Co., Higashi-Hiroshima, Hiroshima 739-0046, Japan, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Hiroshima 734-8553, Japan, and Towa Environment Science Co., Suminoe-ku, Osaka 559-0034, Japan
| | - Shinsaku Naito
- Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan, Preclinical Assessment Department, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima 772-8601, Japan, PhoenixBio, Co., Higashi-Hiroshima, Hiroshima 739-0046, Japan, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Hiroshima 734-8553, Japan, and Towa Environment Science Co., Suminoe-ku, Osaka 559-0034, Japan
| | - Toru Horie
- Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan, Preclinical Assessment Department, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima 772-8601, Japan, PhoenixBio, Co., Higashi-Hiroshima, Hiroshima 739-0046, Japan, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Hiroshima 734-8553, Japan, and Towa Environment Science Co., Suminoe-ku, Osaka 559-0034, Japan
| | - Shigeru Ohta
- Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan, Preclinical Assessment Department, Otsuka Pharmaceutical Factory, Inc., Naruto, Tokushima 772-8601, Japan, PhoenixBio, Co., Higashi-Hiroshima, Hiroshima 739-0046, Japan, Graduate School of Biomedical Sciences, Hiroshima University, Minami-ku, Hiroshima 734-8553, Japan, and Towa Environment Science Co., Suminoe-ku, Osaka 559-0034, Japan
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Keenan J, Elia G, Dunn MJ, Orr DF, Pierscionek BK. Crystallin distribution patterns in concentric layers from toad eye lenses. Proteomics 2009; 9:5340-9. [DOI: 10.1002/pmic.200800986] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
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50
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Eberhardt C, Engelmann S, Kusch H, Albrecht D, Hecker M, Autenrieth IB, Kempf VAJ. Proteomic analysis of the bacterial pathogen Bartonella henselae and identification of immunogenic proteins for serodiagnosis. Proteomics 2009; 9:1967-81. [PMID: 19333998 DOI: 10.1002/pmic.200700670] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Bartonella henselae is a slow growing, fastidious and facultative intracellular pathogen causing cat scratch disease and vasculoproliferative disorders. To date, knowledge about the pathogenicity of this human pathogenic bacterium is limited and, additionally, serodiagnosis still needs further improvement. Here, we investigated the proteome of B. henselae using 2-D SDS-PAGE and MALDI-TOF-MS. We provide a comprehensive 2-D proteome reference map of the whole cell lysate of B. henselae with 431 identified protein spots representing 191 different proteins of which 16 were formerly assigned as hypothetical proteins. To unravel immunoreactive antigens, we applied 2-D SDS-PAGE and subsequent immunoblotting using 33 sera of patients suffering from B. henselae infections. The analysis revealed 79 immunoreactive proteins of which 71 were identified. Setting a threshold of 20% seroreactivity, 11 proteins turned out to be immunodominant antigens potentially useful for an improved Bartonella-specific serodiagnosis. Therefore, we provide for the first time (i) a comprehensive 2-D proteome map of B. henselae for further proteome-based studies focussed on the pathogenicity of B. henselae and (ii) an integrated view into the humoral immune responses targeted against this newly emerged human pathogenic bacterium.
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Affiliation(s)
- Christian Eberhardt
- Institut für Medizinische Mikrobiologie und Hygiene, Klinikum der Eberhard-Karls-Universität, Tübingen, Germany
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