Development of Selective Electrophoresis for Proteins and Peptides Within Proteomes

DDEVELOPMENT OF SELECTIVE ELECTROPHORESIS FOR PROTEINS AND PEPTIDES WITHIN PROTEOMES Thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy LINDA LY School of Biotechnology and Biomolecular Sciences The University of New South Wales August 2008 THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: LY First name: LINDA Other name/s: Abbreviation for degree as given in the University calendar: PhD School: Biotechnology and Biomolecular Sciences Faculty: Science Title: Development of selective electrophoresis for proteins and peptides within proteomes Abstract 350 words maximum: Analysis of complex protein samples is demanding due to the wide dynamic range of expression levels and the limited detection range of technology. Proteomics relies heavily on the development of new fractionation strategies to help reduce complexity, and overcome the technological and biological challenges associated with proteome analysis. Here, the development of a prototype instrument named ‘Microflow MF10’ was explored to enrich for particular classes of proteins. The MF10 was found to have a number of advantages over commercially available fractionation systems. Due to the reduced separation electrode distance, fractionation was rapid, occuring within ~0.125 kVH over 2-6 fractions under native conditions but longer under denaturing conditions. As low as 2 ng peptide could be fractionated with recovery for downstream analysis achievable. The ability to alter protein charge by changing the pH (acidic (pI 3.6) to basic environments (pI 10.4)) allows selection of proteins based on charge/mobility, size, shape, buffer ionic strength, pH and field strength. Proteins <10 kDa are also not routinely analysed because current technology is unable to cater for this region of the proteome. Peptide enrichment using the MF10 was achieved using a 7-protein/peptide standard mix (1-25 kDa), to the 1-5 kDa fraction with simultaneous fractionation of the higher mass protein standards. Plasma was also used to enrich for the peptidome (< 5 kDa) in the presence of the proteome. Enrichment of 73 proteins inclusive of 22 proteins in the 1-25 kDa fraction was achieved compared to a total equivalent of 42 proteins from unfractionated plasma. Rare samples ( 106 cells) from stem cell populations or derived clinically are challenging due to the absolute limits in protein copy number and abundance. CD34+ haematopoietic stem cells and CD4+/CD8+ T-cells were used to develop fractionation methods and elucidate the cell differentiation process. MF10 fractionation and analysis by SDS-PAGE and LC-MS/MS revealed 24 differentially expressed proteins between the 3 cell populations, which may be involved in cell differentiation. To quantify these expression differences, iTRAQ with 2-D LC-MS/MS was applied. This study has highlighted the challenges associated with samples of limited quantity. It has been successful in understanding the effects of various conditions on the electrophoretic mobility of proteins, which in proteomics, has remained largely unexplored. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in 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 property 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 Abstracts International (this is applicable to doctoral theses only). …………………………………………………………… ……………………………………..……………… ……….……………………...…….… Signature Witness Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS ORIGINALITY STATEMENT ‘I 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.’ Signed …………………………………………….............. Date …………/…………/………… L.Ly, University of New South Wales i COPYRIGHT STATEMENT ‘I 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.' Signed …………………………………………….............. Date …………/…………/………… AUTHENTICITY STATEMENT ‘I 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.’ Signed …………………………………………….............. Date …………/…………/………… ii L.Ly, University of New South Wales ACKNOWLEDGEMENTS Thank you to my supervisor Dr. Valerie Wasinger for all her help and guidance with the project and writing. It has been a tough, but rewarding and great learning experience. Thank you also, to my co-supervisor Prof. Michael Guilhaus for his help and advice during the writing process, and for the opportunity to work within an ever-growing and vibrant facility. To everyone at the BMSF, thank you for all your help, guidance, support and friendship. In particular, to Dr. Anne Poljak and Sonia Bustamante for all their assistance around the lab, answering any questions I had, and for the great chats; to Dr. Mark Raftery for all his help and guidance with the mass spectrometry where I have learned a great deal, and for his friendship; and to Lydia Morris for all her help with orders and general lab assistance. You have all made my experience at the BMSF a very enjoyable one. To my fellow PhD mates Alex Roberts, Jasper Pengelly and Joe Codamo, thank you for your help and support. To Lily Ting, for her support and friendship, and for all those seemingly endless (but so much fun) chats about everything and anything. Thank you all for your advice, encouragement and listening to my frustrations throughout it all. Thank you to NuSep for providing the MF10 instrument. Thank you to Dr. Anna Fitzgerald and Dr. Brad Walsh (Minomic), for their help and valuable discussions during parts of this study. Thank you to the Australian Research Council and Australian Postgraduate Award (Industry) for funding and financial support during this work. Finally, thank you to my parents who have loved, supported and cared for me in every way throughout my studies. Thank you to everyone else in my family and friends who have also supported, listened, and encouraged me through tough times. This time, I can definitely say I’m finished. L.Ly, University of New South Wales iii ABSTRACT Analysis of complex protein samples is demanding due to the wide dynamic range of protein expression levels and the limited technology detection range. Proteomics relies heavily on the development of new fractionation strategies to help reduce complexity, and overcome the technological and biological challenges associated with proteome analysis. Electrophoresis is a powerful technique that has been widely used to improve proteome coverage through the partitioning of proteins and peptides in an electric field. Here, the development of a prototype instrument named ‘Microflow MF10’ was explored to enrich for particular classes of proteins. The ability to separate low mass proteins; low volume and concentration samples; and under native and denaturing/reducing conditions, are unique features of this system. The MF10 fractionates proteins and peptides by size and/or charge using pore-restrictive membranes and buffers of defined pH at low volumes. Various conditions were tested using protein and peptide standards. These included: separation time; protein

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