Analysis of secreted proteins from adipocytes: A proteomics approach By Wei Tse Hsu A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science (Research) School of Biotechnology and Biomolecular Sciences University of New South Wales Supervisor: Professor Marc R. Wilkins Co-supervisor: Associate professor Mark Raftery Submitted: 2012 ORIGINALITY STATEMENT ȼI hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ COPYRIGHT STATEMENT ȼI hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' AUTHENTICITY STATEMENT ȼI certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Acknowledgments My thanks go to the many people at UNSW who helped and encouraged me during my time within their department. In particular Mark Raftery, my second supervisor, who provided an insightful view of my work. Also, Ben Herbert, Matt Padula, George Miklos and Graham Vesey, for their willing support in many aspects. I also thank all the Marc Wilkins lab members. Without them the research would not have been possible, especially Ignatius Pang for all his advices on bioinformatic knowledge. I also thank the members of Marc Wilkins lab, especially Simone Li, Melissa Erce, Tim Couttas and Jason Low for their guidance and suggestions. I am particularly indebted to my family members in Taiwan and the best friend Dr. Jerry Wei in University of Sydney and his family members for supporting and encouraging me to pursue this degree. Finally, my biggest thanks go to my supervisor, Marc Wilkins, whose positive, informed, advices and encouraging nature has been an inspiration throughout. Table of contents: ABSTRACT .................................................................................................................................. I LIST OF FIGURES ................................................................................................................... II LIST OF TABLES ....................................................................................................................... V LIST OF ABBREVIATIONS ................................................................................................... VI 1. INTRODUCTION .............................................................................................................. 1 1.1. ADIPOSE TISSUE AND OBESITY ..................................................................................... 1 1.2. PROTEINS SECRETED FROM ADIPOCYTES OR ADIPOSE TISSUES ................................... 2 1.3. EXTRACELLULAR AND INTRACELLULAR SIGNALING COMPONENTS OF ADIPOSE TISSUE OR ADIPOCYTES ......................................................................................................................... 4 1.4. CLASSICAL SECRETORY AND NON-CLASSICAL SECRETORY PATHWAY .......................... 4 1.5. DETECTION OF SECRETED PROTEINS............................................................................ 5 1.6 DETECTION OF PROTEINS USING MASS SPECTROMETRY .............................................. 6 1.7. QUANTITATIVE PROTEOME ANALYSIS .......................................................................... 8 1.7.1. Stable isotope labeling with amino acids in cell culture (SILAC) ........................... 9 1.7.2. Isotope-coded affinity tags (ICAT) .......................................................................... 9 1.7.3. Isotope-coded amine-reactive reagents (iTRAQ) .................................................. 10 1.7.4. Selected or multiple reaction monitoring (SRM/MRM) technology ....................... 11 1.7.5. The absolute quantification method (AQUA) ........................................................ 12 1.7.6. Label-free quantitation .......................................................................................... 15 1.8. PROTEOMICS AND CLINICAL DIAGNOSTICS ................................................................ 15 1.9. HYPOTHESIS AND AIMS .............................................................................................. 17 1.9.1. Hypothesis ............................................................................................................. 17 1.9.2. Aims ....................................................................................................................... 18 2. MATERIALS AND METHODS ..................................................................................... 19 2.1. SAMPLE PREPARATION ............................................................................................... 19 2.1.1. Tissue harvest ........................................................................................................ 19 2.1.2. Cell isolation and culture ...................................................................................... 19 2.2. PROTEIN IDENTIFICATION .......................................................................................... 20 2.2.1. In solution digestion .............................................................................................. 20 2.2.2. Nano-flow LC-MS ................................................................................................. 21 2.3. ANALYSIS OF SECRETED PROTEINS ............................................................................ 22 2.3.1. Proteomic data analysis ........................................................................................ 22 2.3.2. Identification of proteotypic peptides from secreted proteins ............................... 23 2.3.3. Functional analysis of identification proteins ....................................................... 23 3. RESULTS .......................................................................................................................... 24 3.1. IDENTIFICATION OF PROTEINS FROM CONDITIONED MEDIA: CULTURED ADIPOCYTES 25 3.1.1. Subcellular localization of proteins ...................................................................... 27 3.1.2. Functional categorization of proteins ...................................................................... 29 3.1.3. Proteins from adipocytes culture: a summary ....................................................... 34 3.2 IDENTIFICATION OF PROTEINS FROM CONDITIONED MEDIA: ADIPOSE TISSUE EXPLANTS . 38 3.2.1 Subcellular localization of proteins ........................................................................... 40 3.2.2. Functional categorization of proteins ................................................................... 42 3.2.3 Proteins from tissue explants: a summary ............................................................. 49 3.3. COMPARISON BETWEEN TWO TYPES OF CULTURE SYSTEMS ...................................... 52 3.4. QUANTITATIVE ANALYSIS OF CLASSICAL AND NON-CLASSICAL SECRETED PROTEINS 54 3.5. PROTEOTYPIC PEPTIDES DETECTION .......................................................................... 58 3.5.1. Characteristics of peptides identified in this study ............................................... 58 3.5.2. Theoretical proteotypic peptides ........................................................................... 60 3.5.3. Experimental proteotypic peptide detection .......................................................... 63 3.5.4. Selected reaction monitoring (SRM) method prediction ....................................... 67 4. DISCUSSION ................................................................................................................... 71 4.1. GEL AND LABEL-FREE SECRETOMICS STRATEGIES IN SERUM-FREE ADIPOCYTES AND ADIPOSE EXPLANTS ................................................................................................................. 71 4.2. NEW PROTEINS DETECTED IN THIS STUDY ................................................................. 80 4.3. CLINICAL APPLICATION OF OUR RESULTS