UNIVERSITÀ DEGLI STUDI DI PADOVA DIPARTIMENTO DI MEDICINA MOLECOLARE SCUOLA DI DOTTORATO DI RICERCA IN: BIOMEDICINA CICLO XXV SETTING UP OF AN INNOVATIVE PROCEDURE FOR REDOX PROTEOMICS AND ITS APPLICATION FOR DEFINITION OF THE REDOX STATUS OF CELLS WITH HIGH METASTATIC POTENTIAL Direttore della Scuola: Ch.mo Prof. Giorgio Palù Supervisore: Ch.mo Prof. Fulvio Ursini Dottorando: Mattia Zaccarin ABBREVIATIONS 2D Two-dimensional 5-IAF 5-(Iodoacetamido)fluorescein Ab Antibody ACN Acetonitrile APS Ammonium persulfate BSA Bovine serum albumine CCB Coomassie Colloidal Blue CID Collision induced dissociation CSCs Cancer Stem Cells DIGE Differential in Gel Electrophoresis DOC Sodium deoxycholate DTNB 3,3’-dithio-bis(6-nitrobenzoic acid) DTT Dithiothreithol EDTA Ethylenediaminetetraacetic acid EMT Epithelial to mesenchymal transition ESI Electrospray ionization FA Formic acid G6PDH Glucose-6-phosphate dehydrogenase GSH Reduced glutathione HPDP N-[6-(Biotinamido)hexyl]-3´-(2´-pyridyldithio)propionamide HRP Horseradish peroxidase IAA Iodoacetic acid IAM Iodoacetamide LC Liquid chromatography m/z Mass to charge ratio MS Mass Spectrometry MS/MS Tandem mass spectrometry MW Molecular weight NEM N-ethylmaleimide PAGE Polyacrylamide electrophoresis PBS Phosphate buffered saline PEP Posterior Error Probability PPP Pentose phosphate pathway Q-TOF Quarupole – Time of flight RNS Reactive nitrogen species ROS Reactive oxygen species RPC Reverse phase chromatography Rt Retention time SDS Sodium dodecyl sulphate TCA 2,2,2-Trichloroacetic acid TCEP Tris(2-carboxyethyl)phosphine TEMED Tetramethylethylenediamine TrisHCl Trishydroxymethylaminomethane hydrochloride Wb Western blotting 1 CONTENTS ABBREVIATIONS.................................................................................................................... 1 1. ABSTRACT ........................................................................................................................... 5 RIASSUNTO ............................................................................................................................. 6 2. INTRODUCTION ................................................................................................................. 8 2.1 REDOX STATUS OF PROTEINS .................................................................................................... 8 2.1.1 Reactivity of Cysteine residues ........................................................................................ 9 Disulfides........................................................................................................................................... 10 Thiyl radical ...................................................................................................................................... 11 Sulfenic acid ...................................................................................................................................... 12 Reaction of thiols with hydrogen peroxide ........................................................................................ 13 2.1.2 Redox switches ............................................................................................................... 14 2.1.3 Structural and functional disulfides ............................................................................... 17 2.2 OXIDATION RATE AND REDUCTION RATE ............................................................................... 20 Specificity and reversibility of redox signalling ................................................................................ 20 Dynamic equilibrium: oxidation and reduction rate .......................................................................... 21 2.2.1 Electrophiles and nucleophiles formation ..................................................................... 23 Sources of biologically relevant ROS ................................................................................................ 24 Antioxidant defense mechanisms ...................................................................................................... 25 2.2.2 Nrf2-Keap1 feedback loop ............................................................................................. 27 Nrf2 in cancer prevention and promotion: dual roles of Nrf2 ............................................................ 28 2.3 EVENTS RELATED TO MALIGNANCY ARE REDOX REGULATED................................................. 29 2.3.1 Oxidation ....................................................................................................................... 31 DNA and lipids .................................................................................................................................. 31 Proteins .............................................................................................................................................. 32 Non receptor tyrosine kinases (PTKs): Src ........................................................................................ 33 Rat Sarcoma genes product (Ras) and the Raf/MEK/ERK pathway ................................................. 33 PI3K/AKT pathway and protein tyrosine phosphatases: PTP1B and PTEN ..................................... 34 2.3.2 Reduction ....................................................................................................................... 35 2.3.3 Localization ................................................................................................................... 37 GSH/GSSG redox potential in cellular compartments ....................................................................... 37 Redox regulation of cell migration and adhesion .............................................................................. 38 2.4 METHODOLOGICAL APPROACH: TRADITIONAL AND INNOVATIVE METHODS FOR DISULFIDE BONDS ASSESSMENT..................................................................................................................... 40 2.4.1 Disulfide proteome of complex samples......................................................................... 42 3. MATHERIALS AND METHODS ...................................................................................... 44 3.1 CELL CULTURES ..................................................................................................................... 44 3.2 SUB-CELLULAR FRACTION ENRICHMENT ................................................................................ 44 3.2.1 Protein assay ................................................................................................................. 45 3.2.2 DTNB assay ................................................................................................................... 45 3.3 DIFFERENTIAL “REDOX” LABELLING ...................................................................................... 46 Alkylation of free thiols ..................................................................................................................... 46 Reduction of oxidized thiols .............................................................................................................. 46 HPDP Labelling................................................................................................................................. 46 Enrichment of labelled proteins ......................................................................................................... 46 Alkylation of formerly oxidized thiols .............................................................................................. 47 3.3.1 HPDP assay ................................................................................................................... 48 2 3.3.2 Electrophoresis .............................................................................................................. 48 3.3.3 Western blotting............................................................................................................. 48 3.4 SAMPLE PREPARATION FOR MS ANALYSIS ............................................................................. 49 3.5 Q-TOF ANALYSIS .................................................................................................................. 49 3.6 G6PDH ENZYMATIC ACTIVITY MEASUREMENT ..................................................................... 50 3.7 DATA ANALYSIS..................................................................................................................... 51 3.7.1 Input data ...................................................................................................................... 52 3.7.2 Identification ................................................................................................................. 52 3.7.3 Quantification ................................................................................................................ 54 3.7.4 Results filtering and score ranking ................................................................................ 55 4. RESULTS............................................................................................................................. 57 4.1 DEVELOPMENT OF LABEL FREE METHODOLOGY TO CHARACTERIZE AND DIFFERENTIALLY QUANTIFY OXIDATIVELY MODIFIED PROTEINS IN COMPLEX SAMPLE ............................................ 57 4.1.1 Differential redox labelling ........................................................................................... 57 4.1.1.1 Trapping of the native redox state of thiols ..........................................................................