Development of novel mass spectrometry-based approaches for searching for low-mass tyrosinase inhibitors in complex mixtures Aleksander Salwiński To cite this version: Aleksander Salwiński. Development of novel mass spectrometry-based approaches for searching for low-mass tyrosinase inhibitors in complex mixtures. Other. Université d’Orléans, 2014. English. NNT : 2014ORLE2013. tel-01223001 HAL Id: tel-01223001 https://tel.archives-ouvertes.fr/tel-01223001 Submitted on 1 Nov 2015 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. 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UNIVERSITÉ D’ORLÉANS ÉCOLE DOCTORALE SANTE, SCIENCES BIOLOGIQUES ET CHIMIE DU VIVANT Institut de Chimie Organique et Analytique THÈSE présentée par : Aleksander SALWIŃSKI soutenue le : 24 avril 2014 pour obtenir le grade de : Docteur de l’université d’Orléans Discipline/ Spécialité : Chimie Development of novel mass spectrometry-based approaches for searching for low-mass tyrosinase inhibitors in complex mixtures THÈSE dirigée par : Benoît MAUNIT Professeur – Université d'Orléans Raphaël DÉLÉPEE Professeur – Université de Caen Basse Normandie RAPPORTEURS : Francesc Xavier AVILÉS Professeur – Université Autonome de Barcelone Benjamin CARBONNIER Professeur – Université Paris-Est Créteil Val de Marne JURY : Francesc Xavier AVILÉS Professeur – Université Autonome de Barcelone Benjamin CARBONNIER Professeur – Université Paris-Est Créteil Val de Marne Patrice ANDRÉ Président de BOTANICOSM'ETHIC Claire DEMESMAY-GUILHIN Professeur – Université Claude Bernard Lyon 1 Paul HANNEWALD Docteur – Directeur énéral Ad oint de 'Plant Advanced Technologies' Raphaël DÉLÉPEE Professeur – Université de Caen Basse Normandie Benoît MAUNIT Professeur – Université d'Orléans CONFIDENTIAL Acknowledgements This thesis was conducted in the Institute of Organic and Analytical Chemistry (ICOA) in Orleans, France. I would like to thank Professor Olivier Martin, director of ICOA, and Professor Claire Elfakir, head of the laboratory of analytical chemistry, for Their kind welcome in the Institute. I would like to thank my scientific supervisors: Professor Benoît Manunit and Professor Raphaël Delépée for giving me the opportunity to conduct the PhD research project in Their scientific team. I would like to thank each of You for Your support, assistance and suggestions. I would also like to thank Doctor David Da Silva for His help in conducting various FA and ENALDI-MS experiments in the final stage of my work and numerous precious advices and remarks concerning the final form of the present manuscript. I would like to thank Professor Francesc Xavier Avilés (Autonomous University of Barcelona) and Professor Benjamin Carbonnier (University Paris-Est Créteil) for giving me a honour to review this work and to become Reporters of my Thesis manuscript. I thank Professor Claire Demesmay-Guilhin, Doctor Paul Hannewald and Mr Patrice André for giving me a honour to join the Jury in the role of Examiners. I thank Doctor Cyril Colas, Doctor Emilie Destandau, Sandrine Zubrzycki and Laëtitia Fougere for Their inestimable help and assistande during my work in the laboratory. A number of other people deserve many thanks for Their presence in my everyday laboratory work. I would like thank them for their enthusiasm and kindness: Syame, Hanane, Ludivine, Manabu-san, Aleksandra, Nathaly, Hala, Reine, Ozkan and Kinga. I am thankful to the Conseil régional du Centre for financial support. I would like to thank Mr. Patrice André, director of the Ethnobotany Innovation Department of LVMH Recherche, for providing us with the samples of plant extract and glabridin. TABLE OF CONTENTS List of abbreviations GENERAL INTRODUCTION Chapter I – Introduction I. Preface......................................................................................................................................... 1 I.1. Skin hyperpigmentation: a general outlook............................................................................. 2 I.2. Tyrosinase - the main enzyme responsible for skin pigmentation............................................. 3 I.2.1. The pathway of melanin biosynthesis enzymatic conversion of L-Tyr and L-DOPA................ 6 I.2.2. General introduction to enzyme kinetics and types of enzyme inhibition.................................. 7 I.2.2.1. An outlook of tyrosinase inhibitors......................................................................................... 8 II. High-throughput screening as a tool for finding biologically active substances............................ 1 4 II.1. Affinity-based screening......................................................................................................... 15 II.1.1. Affinity chromatography.......................................................................................................... 16 II.1.2. Surface plasmon resonance coupled with HPLC/MS................................................................. 17 II.1.3. Ultrafiltration and related techniques...................................................................................... 19 II.1.4. Direct detection of protein-ligand complexes........................................................................... 20 II.1.5. Intensity ion fading MS............................................................................................................. 22 II.2. On-line enzymatic activity-based screening............................................................................. 23 II.3. A short overview of high-throughput screening methods developed in present work.............. 25 III. Mass spectrometry..................................................................................................................... 25 III.1. Introduction........................................................................................................................... 25 III.2. Mass spectrometer with electrospray ion source.................................................................... 26 III.2.1 Electrospray ion source............................................................................................................. 26 III.2.2. Collision-induced dissociation and its application in metabolite identification....................... 28 III.2.3. An outlook of Bruker maXisTM UHR-Qq-TOF spectrometer...................................................... 30 III.3. Matrix-assisted laser desorption/ionisation and related techniques........................................ 34 IV. The strategies for protein immobilisation................................................................................... 39 IV.1. Non-covalent protein immobilisation..................................................................................... 39 IV.1.1. Immobilisation based on specific protein-ligand interactions................................................. 39 IV.1.2. Immobilised artificial membranes........................................................................................... 41 IV.1.3. Enzyme immobilisation by entrapment within a polymer network........................................ 41 IV.2. Covalent protein immobilisation............................................................................................ 41 IV.2.1. General outlook....................................................................................................................... 41 IV.2.2. Impact of protein immobilisation on its kinetic parameters: KM and Vmax............................... 44 Chapter II – Frontal affinity chromatography I. Frontal affinity chromatography: from theory to practice............................................................. 45 I.1. An introduction to frontal affinity chromatography................................................................. 45 I.1.1. The historical background of frontal affinity chromatography.................................................. 45 I.1.2 The principle and mathematical description of frontal analysis................................................. 45 I.1.2.1. A 'single infusion' approach.................................................................................................... 46 I.1.2.2. Staircase frontal analysis........................................................................................................ 48 I.2. Selection of appropriate detection method.............................................................................. 51 I.3. Non-specific interactions of analyte with the stationary phases. Selection of appropriate immobilisation support.......................................................................................................... 52 I.4. The pathways of protein immobilisation commonly applied for frontal affinity chromatography............................................................................................................................ 53 II. Stationary phases for FAC: development and application of FAC for searching for the inhibitors of tyrosinase and trypsin.........................................................................................................