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Proteomic Profiling of Phosphoproteins and Secreted

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Proteomic Profiling of Phosphoproteins and Secreted Proteomic profiling of phosphoproteins and secreted proteins from mammalian cell lines in order to gain insights into factors affecting cellular growth and recombinant protein production. A thesis submitted for the degree of PhD by Martin Power, BSc. April 2015 The experimental work in this thesis was performed under the supervision of Dr. Paula Meleady & Prof. Martin Clynes School of Biotechnology Dublin City University I hereby certify that this material, which I now submit for assessment on the programme of study leading to the award of PhD is entirely my own work, that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge breach any law of copyright, and has not been taken from the work of others save and to the extent that such work has been cited and acknowledged within the text of my work. Signed: ____________________________ ID No.: __________________ Date:_________________ This thesis is dedicated to my mother, Doris and the memory of my father, Frank. Acknowledgements Firstly I would like to thank my supervisor Dr. Paula Meleady for taking me on and guiding me through the PhD process with endless positivity, encouragement and support. I would also like to thank Prof. Martin Clynes for giving me the opportunity to carry out my work in the National Institute for Cellular Biotechnology. I am sincerely grateful for your unwavering support and guidance over the course of the long journey towards my PhD. A special thank you to Michael Henry for all his help with the mass spec work (of which there was no shortage in my project!). Thank you for all the time you gave me and your endless patience. Thank you to Dr. Annemarie Larkin for all her help with the hybridoma work and to Dr. Colin Clarke for his help with the bioinformatics. Also thank you to Carol, Yvonne and Mairead for all their help in the background to keep the show on the road. A special thank you to John, Kate and Bernadette in NIBRT for all the support they showed me during the final stages of my PhD. Thank you to Noelia, Shane, Mark, Paul, Alan, Gemma, Erica, Deidre and Damian and all my friends in the NICB for making it such a great place to come to work every day. Finally to my mum, Doris, thank you for being so patient for so long. Now, I’m finally finished! Table of Contents Abstract 1 1.0 Introduction 3 1.1 Chinese Hamster Ovary cells 3 1.2 Temperature shift 7 1.2.1 Mechanisms controlling Cell growth rates in temperature shifted CHO cells 7 1.2.2 Mechanisms controlling specific productivity in temperature shifted CHO cells 8 1.2.3 Proteomic profiling of temperature shifted CHO cells 12 1.3 Proteomic profiling of desirable cell phenotypes 13 1.4 Host cell proteins (HCPs) 17 1.4.1 Proteases as part of the HCP matrix 19 1.4.2 Growth Factors and Conditioned Media 21 1.5 Introduction to Proteomics 24 1.5.1 Proteomic sample preparation 24 1.5.2 Sample separation 25 1.5.2.1 Protein separation by 2D-PAGE 26 1.5.2.2 Protein separation by Liquid chromatography 27 1.5.3 Mass spectrometry for protein identification and characterisation 28 1.5.3.1 Sample preparation for Mass Spectrometry 28 1.5.3.2 Nanoflow Liquid Chromatography coupled to Mass Spectrometers 29 1.5.3.3 The basic components of a mass spectrometer 30 1.5.3.4 Determining the peptide sequence from mass spectrometry data 34 1.5.4 Mass spectrometry data output 36 1.5.5 Bioinformatic analysis of mass spectral data 36 1.5.5.1 CHO specific proteomic databases 38 1.5.6 Post Translational Modifications (PTM) Analysis using Mass Spectrometry 39 1.5.7 Identification of phosphopeptides by mass spectrometry 40 1.5.8 Phosphoproteomic databases 43 1.5.9 Quantitative label free LC/MS 45 1.6 Analysis of protein phosphorylation 47 1.6.1 Protein arrays to study phosphorylation 50 1.6.2 Staining of phosphoproteins for gel based approaches 51 1.6.3 Chromatography for the enrichment of phosphoproteins and phosphopeptides 52 1.6.3.1 Titanium dioxide phosphopeptide enrichment 53 1.6.3.2 IMAC for phosphopeptide enrichment 55 1.6.3.3 Strong cation exchange (SCX) 56 Aims 58 2.0 Materials & Method 61 2.1 Preparation for Cell Culture 61 2.1.1 Ultrapure water 61 2.1.2 Glassware 61 2.1.3 Sterilisation 61 i 2.1.4 Subculturing of cell lines suspension cells 61 2.1.5 Assessment of cell number and viability 63 2.1.6 Cryopreservation of cells 63 2.1.7 Thawing of cryopreserved cells 63 2.1.8 Sterility checks 64 2.1.9 Mycoplasma testing 64 2.2 Protein Quantification 64 2.2.1 Bradford Assay 64 2.2.2 ELISA Assay 65 2.2.3 SEAP Assay 65 2.3 Proteomic Sample Collection and Preparation 66 2.3.1 Cell Lysate Sample Preparation 66 2.3.2 Conditioned Media Collection 67 2.3.3 Conditioned Media Sample Preparation 67 2.3.4 Sample clean-up for Mass Spectral Analysis 67 2.3.5 In solution digestion for preparation of conditioned media samples 67 2.3.6 In solution digestion for preparation of whole cell lysate samples 68 2.3.7 Peptide sample clean-up using C18 spin column 68 2.3.8 Peptide sample clean-up using Graphite spin column 69 2.4 Proteomic Enrichment Methods 69 2.4.1 Phosphoprotein enrichment Pierce 69 2.4.2 Phosphoprotein Label Free 70 2.4.3 Gallium Phosphopeptide enrichment 70 2.4.4 TiO2 Phosphopeptide enrichment 70 2.4.5 Iron Phosphopeptide enrichment 70 2.4.6 LC/MS analysis 71 2.4.7 Mass Spectrometry Protein Identifications 72 2.4.8 Label-free LC-MS data analysis 73 2.5 Other Proteomic Techniques 74 2.5.1 Gel electrophoresis 74 2.5.2 Western blotting 74 2.5.3 Enhanced chemiluminescence (ECL) detection 75 2.5.4 MAP Kinase array 76 2.5.5 Depletion of IgG from conditioned media collected from DP12 cell line 77 3.0 Host Cell Protein Analysis 79 3.1 Characterisation of the host cell protein profile of a non-producing hybridoma cell line 80 3.1.1 Growth profiling of non-producing hybridoma cell line 80 3.1.2 Quantitative label free LC/MS analysis of conditioned media from a non-producing hybridoma cell line 82 3.1.3 Data analysis of differentially regulated proteins identified by label free LC/MS profiling of conditioned media from a hybridoma cell line 82 3.1.3.1 Differential regulation of HCPs 24hrs vs 96hrs at 37˚C 83 3.1.3.2 Differential regulation of HCPs 24hrs vs 96hrs at 31˚C 85 3.1.3.3 Differential regulation of HCPs at 24hrs 37˚C vs 31˚C 87 3.1.3.4 Differential regulation of HCPs at 96hrs 37˚C vs 31˚C 89 ii 3.1.3.5 Comparison of differentially regulated HCPs between 24hrs and 96hrs post temperature shift at 37˚C and 31˚C in a non-producing hybridoma cell line 91 3.1.4 Qualitative analysis of proteins identified by label free LC/MS profiling of conditioned media from a non-producing hybridoma cell line 94 3.1.5 Summary 96 3.2 Characterisation of the Conditional Medium host cell protein profile of a non-producing CHO K1 cell line 98 3.2.1 Growth profiling of non-producing CHO K1 cell line 98 3.2.2 Quantitative Label free LC/MS analysis of conditioned media from a CHO K1 non-producing cell line 100 3.2.3 Data analysis of differentially regulated proteins identifi ed by quantitative label free LC/MS profiling of conditioned media from a CHO K1 non-producing cell line 101 3.2.3.1 Differential regulation of HCPs 24hrs vs 120hrs at 37˚C 102 3.2.3.2 Differential regulation of HCPs 24hrs vs 120hrs at 31˚C 105 3.2.3.3 Differential regulation of HCPs at 24hrs 37˚C vs 31˚C 108 3.2.3.4 Differential regulation of HCPs at 120hrs 37˚C vs 31˚C 111 3.2.3.5 Pearson correlation analysis of differentially regulated HCPs over 24, 72, 96 and 120 hours at 37˚C 114 3.2.3.6 Pearson correlation analysis of differentially regulated HCPs over 24, 72, 96 and 120 hours at 31˚C 116 3.2.3.7 Comparison of differentially regulated HCPs over time in culture at 37˚C and 31˚C in a non-producing CHO K1 cell line 118 3.2.3.8 Comparison of quantitative label-free LC/MS CHO-K1 HCP data normalised to cell number compared to normalisation by protein concentration 125 3.2.4 Bioinformatic analysis of differentially regulated protein lists 126 3.2.5 Qualitative analysis of proteins identified by quantitative label free LC/MS profiling of conditioned media from a non-producing CHO K1 cell line 132 3.2.5.1 Qualitative comparison of HCPs identified in temperature shifted and non-temperature shifted CHO K1 cultures 133 3.2.6 Summary 135 3.3 Characterisation of the host cell protein profile of an IgG producing DP12 cell line 136 3.3.1 Growth curves at 31˚C and 37˚C of IgG producing DP12 cell line 136 3.3.2 IgG depletion of conditioned media from IgG producing DP12 CHO cell line 138 3.3.3 Quantitative label free LC/MS analysis of conditioned media from a DP12 CHO cell line 140 3.3.4 Data analysis of differentially regulated proteins identified by quantitative label free LC/MS profiling of conditioned media from a DP12 CHO cell line 141 3.
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