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Single Molecule Protein Detection for Proteomic Profiling Simon Leclerc Single molecule protein detection for proteomic profiling Simon Leclerc To cite this version: Simon Leclerc. Single molecule protein detection for proteomic profiling. Genomics [q-bio.GN]. Uni- versité de Strasbourg, 2018. English. NNT : 2018STRAJ044. tel-02124182 HAL Id: tel-02124182 https://tel.archives-ouvertes.fr/tel-02124182 Submitted on 9 May 2019 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. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE des Sciences de la Vie et de la Santé UMR 1121 du CNRS THÈSE présentée par : Simon LECLERC soutenue le : 24 Août 2018 pour obtenir le grade de : Docteur de l’université de Strasbourg Discipline/ Spécialité : Biologie moléculaire Visualisation de protéines individuelles pour la quantification à haute sensibilité du lysat cellulaire THÈSE dirigée par : Dr Arntz Youri................................................................Docteur, Université de Strasbourg Dr Taniguchi Yuichi...................................................................................Docteur, RIKEN RAPPORTEURS : Dr Cuisinier Frederic ...............................................Professeur, Université Montpellier 1 Dr Csilla Gergely.......................................................Professeur, Université Montpellier 2 AUTRES MEMBRES DU JURY : Dr Vautier Dominique................................................IR inserm, Université de Strasbourg Acknowledgment Firstly, I would like to express my sincere gratitude to my advisers Dr Taniguchi Yuichi and Dr Arntz Youri for the continuous support of my Ph.D study, administrative troubles and related research, for their patience, motivation, and knowledge. Their guidance helped me in all the time of research and writing of my article and this thesis. I could not have imagined having better advisers and mentors for my Ph.D study. Besides my advisers, I would like to thank the rest of my thesis committee: Prof. Cuisinier Frederic, Prof. Csilla Gergely, and Dr. Vautier Dominique, for their insightful comments and encouragement, but also for the hard question which incented me to widen my research from various perspectives. My sincere thanks also goes to Dr. YanagidaToshio who provided me an opportunity to join their team as intern, and who gave access to the laboratory and research facilities. Without they precious support it would not be possible to conduct this research. I thank my fellow labmates Vipin and Arno for the stimulating discussions, how to recreate the world and for all the fun we have had in the last four years. I also like to thanks the other staff and labmates from QbiC, RIKEN, but also the staff of Strasbourg University Last but not the least, I would like to thank my loving wife for the continuous support during the thesis and article redaction, and my family, parents and sisters for supporting me spiritually throughout writing this thesis and my life in general. Table of content Index of Tables.......................................................................................................................1 Index of Figure.......................................................................................................................2 Index of Annex........................................................................................................................3 Abbreviations table.................................................................................................................4 Introduction.............................................................................................................................6 Biology : what a noisy process!..........................................................................................5 Stochastic precision...........................................................................................................7 New from old : single protein detection..............................................................................8 Achieving ultimate sensitivity...........................................................................................13 Part I : Determination of the labeling efficiency.....................................................................15 Clearing the way for single molecule observation............................................................16 What means homogeneity at single molecule..................................................................17 Control experiment and robustness test...........................................................................24 Improving the labeling......................................................................................................29 Proteome scale labeling...................................................................................................33 Possible improvements....................................................................................................41 Conclusion.......................................................................................................................42 Part II : Proteome profiling at single molecule level..............................................................45 Capillary electrophoresis for profiling...............................................................................44 Profiling on thin SDS-PAGE.............................................................................................51 Discussion and Conclusion...................................................................................................61 Discussion............................................................................................................................62 Résumé étendu en Français.................................................................................................71 Introduction......................................................................................................................71 Résultats..........................................................................................................................72 Discussion.......................................................................................................................81 Conclusion.......................................................................................................................83 References...........................................................................................................................86 Index of Tables-Index of Tables Index of Tables Table 1: Single cell analysis tools for protein studies..............................................................8 Table 2: Labeling buffer composition at each condition.........................................................30 Table 3: Top 10 of protein abundance in Hela cell................................................................35 1 Index of Figure-Index of Figure Index of Figure Central dogma in molecular biology........................................................................................5 Stochastic gene expression process......................................................................................7 Single protein detection by deep UV fluorescence.................................................................9 Single protein detection by nanopore.....................................................................................9 Single molecule array principle.............................................................................................10 Proximity ligation assay........................................................................................................11 Single protein counting in microfluidic...................................................................................12 Fluorescent protein counting................................................................................................13 Cy3 and different reactive functions......................................................................................15 The effect of labeling homogeneity on protein number counting...........................................18 Assay to measure the labeling homogeneity........................................................................19 Microscope setting for single molecule visualization.............................................................21 Controls for avidin-biotin reaction.........................................................................................22 Evaluation of labeling homogeneity......................................................................................23 Single molecule photobleaching experiment.........................................................................24 Pixel values from the background and detected spots..........................................................25 Insensitivity of the sample protein amount on the output spot number in the assay system. 26 Effect of concentration ratio of the dyes to proteins during the labeling reaction on the LO..27 Effect of the image number on the LO accuracy...................................................................28
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