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High pressure liquid chromatography for functional and structural proteomics analysis

English title High pressure liquid chromatography for functional and structural proteomics analysis
Applicant Koppenol Willem H.
Number 145015
Funding scheme R'EQUIP
Research institution Laboratorium für Anorganische Chemie Departement für Chemie und Angewandte Biowis ETH Zürich
Institution of higher education ETH Zurich - ETHZ
Main discipline Inorganic Chemistry
Start/End 01.01.2013 - 31.12.2013
Approved amount 47'000.00
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All Disciplines (2)

Discipline
Inorganic Chemistry
Biochemistry

Keywords (14)

protein oxidation; alpha-A-crystallin; alpha-B-crystallin; alpha-synuclein; cataract; oxidative stress; protein misfolding; heat-shock protein; gamma radiolysis; photolysis; electron transfer; radical reaction; product analysis; high pressure liquid chromatography

Lay Summary (German)

Lead
Die Proteine der Augenlinsen alpha-A- und alpha-B-Crystallins sind Angriffsfläche für Licht und Luft, was zu massivem oxidativem Stress führt. Die Oxidation in vivo von Crystallins trägt zur Entstehung von grauem Star bei. Ausserhalb der Augenlinse funktionieren alpha-B-Crystallin und alpha-Synuclein als so-genannte Chaperon Proteine, die andere Proteine von Fehlfaltung schützen. Bei alpha-B-Crystallin und alpha-Synclein kann es in Reaktion auf oxidativen Stress auch zu Fehlfaltung kommen.
Lay summary

Inhalt und Ziel des Forschungsprojekts

Wir untersuchen die Mechanismen, wie Radikale Schädigungen an Aminosäuren, Peptiden und Proteinen verursachen und wie Antioxidantia diese schützen und reparieren. Im Labor verwenden wir Gamma-Strahlen, UV-Licht und Fenton-Chemie als oxidativen Stress; nach der Schädigung analysieren wir die molekularen Veränderungen. Hochdruckflüssigkeitschromatographie (HPLC) ist das Forschungswerkzeug, das wir anwenden, um die verschiedenen Produkte der oxidativen Reaktionen zu finden und zu trennen. Für eine eindeutige Identifikation der einzelnen Produkte benötigen wir HPLC-integrierte Massenspektrometrie, Diodenarray,  und Fluoreszenz Detektoren.

Wissenschaftlicher und gesellschaftlicher Kontext des Forschungsprojekts

Das neue HPLC-Gerät wird im Zusammenhang mit SNF Projekt Nr. 200020_135106, "In vivo targets of protein oxidation", verwendet, um die Produkte der Proteinoxidationsreaktionen zu analysieren. Zudem wird das Gerät sowohl für die Analyse von Reaktionen mit Cytochrome P-450 im Rahmen des SNF Sinergia Projekts Nr. CRSII2_127547/1, "Innovative enabling micro-nano-bio-technologies for implantable systems in molecular medicine and personalized therapy" als auch für Untersuchungen von oxidativ beschädigten Brennstoffzelle-Membranen in Zusammenhang mit ETH Projekt Nr. 02 11-1, "Probing the Degradation of Fuel Cell Membranes by Time-Resolved Spectroscopy" eingesetzt.

Direct link to Lay Summary Last update: 22.11.2012

Lay Summary (English)

Lead
The eye lens proteins, alpha-A- und alpha-B-crystallins, are front-line targets for oxidatve stress from exposure to light and air. In vivo oxidation of crystallins plays a role in cataract formation. In extra-lens tissues, alpha-B-crystallin und alpha-synucleinfunctions as so-called chaperone proteins to protect other proteins from misfolding. Oxidative stress can can also cause misfolding in alpha-B-Crystallin und alpha-Synuclein.
Lay summary

Content and aims of the research project

We investigate mechanisms of radical damage to amino acids, peptides, and proteins and how antioxidants protect against and repair that damage. In the laboratory, we use gamma radiation, UV light, and Fenton chemistry as sources of oxidative stress; post reaction, we analyze molecular changes in the damaged molecules. High pressure liquid chramatography (HPLC) is the research tool we use to separate the various products of oxidative reactions for analysis. For unambiguous identification of the individual products, we need HPLC-integrated mass spectrometry, diode-array UV/vis, and fluorescence detectors.

  

Scientific and social context of the research project

The new HPLC instrument will be used  to analyze products of protein oxidation as part of SNF Project No. 200020_135106, "In vivo targets of protein oxidation". Further, the instrument will be used to analyze products of Cytochrome P-450 reactions described in the SNF Sinergia Project No. CRSII2_127547/1, "Innovativeenabling micro-nano-bio-technologies for implantable systems in molecularmedicine and personalized therapy" as well as for investigation of oxidative damage to fuel cell membranes as part of ETH Project No. 02 11-1,"Probing the Degradation of Fuel Cell Membranes by Time-Resolved Spectroscopy".

Direct link to Lay Summary Last update: 22.11.2012

Responsible applicant and co-applicants

Publications

Publication
Redox properties and activity of iron-citrate complexes: Evidence for redox cycling.
Adam Fatima I. Bounds Patricia L. Koppenol Willem H. (2015), Redox properties and activity of iron-citrate complexes: Evidence for redox cycling., in Chemical Research in Toxicology, 28(4), 604-614.

Collaboration

Group / person Country
Types of collaboration
Prof. C. Schöneich, Pharmaceutical Chemistry, University of Kansas United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
University of Zürich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Prof. U. von Mandach, Obstetrics and Gynecology, University of Zurich Hospital Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Dr. L. Gubler, Electrochemistry Laboratory, PSI Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Fall Meeting of the Swiss Chemical Society Poster Modulation of tyrosine oxidation by methionine in α-synuclein 06.09.2013 Lausanne, Switzerland Koppenol Willem H.;
18th International Conference on Cytochrome P450: Biochemistry, Biophysics and Biotechnology Talk given at a conference Conservative Point Mutations in Cyp3a4 Elicit Cooperative Kinetics in the Reaction with Carbamazepine 18.06.2013 Seattle, Washington USA, United States of America Koppenol Willem H.;
18th International Conference on Cytochrome P450: Biochemistry, Biophysics and Biotechnology Poster Conservative Point Mutations in Cyp3a4 Elicit Cooperative Kinetics in the Reaction with Carbamazepine 18.06.2013 Seattle, Washington USA, United States of America Koppenol Willem H.;
European Young Investigator Conference 2013, EYIC 2013 Talk given at a conference Degradation of polystyrenesulfonic acid caused by gamma irradiation 07.01.2013 Slubice, Poland Koppenol Willem H.;


Associated projects

Number Title Start Funding scheme
135106 Mechanisms of Oxygen Toxicity: In Vivo Targets of Protein Oxidation 01.04.2011 Project funding
127547 Innovative Enabling Micro-Nano-Bio-technologies for Implantable systems in molecular medicine and personalized therapy 01.01.2010 Sinergia

Abstract

We investigate radical mediated oxidation of amino acids, peptides, and proteins to elucidate mechanisms of damage, protection and repair. In vivo, eye lens crystallins are front-line targets of considerable oxidative stress because of exposure to light and air; in extra-lens tissues, alpha-B-crystallin and alpha-synuclein undergo misfolding, leading to formation of amyloid-like structures, in response to oxidative stress. We study the antioxidant protection and repair of crystallins and alpha-synuclein during and after exposure to oxidizing and reducing species generated by ionizing and UV radiation and by Fenton chemistry, for which we analyze post-translational modifications of target proteins caused by oxidative stress. High performance liquid chromatography (HPLC) is the research tool we use to separate the multiple products we find from oxidative reactions; mass spectrometry, diode-array, and fluorescence are the modes of detection we need to unambiguously identify the individual products. We request funding to replace our existing HPLC system. The existing system is 17 years old and has been written off. It consists of Hewlett Packard series 1050 and Agilent 1100 modules that are no longer supported by Agilent service; replacement consumable parts are difficult to obtain. The system is controlled by computer via an un-upgradable interface, by software that runs on Windows NT (does not support even Windows XP).The instrument we plan to purchase will consist of quaternary solvent delivery and autosampler components, and mass spectrometry, diode array and fluorescence detectors. The new state-of-the-art system will be able to perform analyses in less time and will consume less organic modifier, thereby producing less environmentally unfriendly organic waste.In addition to the SNF "In vivo targets of protein oxidation" project (200020_135106), we will use the new HPLC for analyses related to the SNF Sinergia project "Innovative enabling micro-nano-bio-technologies for implantable systems in molecular medicine and personalized therapy" (Nr. CRSII2_127547/1) and ETH projects The instrument will be transferred in ca. 2 years to Prof. Copéret upon Prof. Koppenol's retirement.
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