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Antibody-Based Subcellular Localization of the Human Proteome

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Antibody-Based Subcellular Localization of the Human Proteome Antibody-based subcellular localization of the human proteome Marie Skogs Licentiate Thesis KTH - Royal Institute of Technology School of Biotechnology Stockholm, Sweden 2016 © Marie Skogs 2016 KTH – Royal Institute of Technology School of Biotechnology Division of Proteomics and Nanobiotechnology Science for Life Laboratory Tomtebodavägen 23A Solna Sweden TRITA-BIO Report 2016:13 ISSN 1654-2312 ISBN 978-91-7729-010-0 Cover illustration by Ina Schuppe Koistinen www.inasakvareller.se Other illustrations by Marie Skogs unless otherwise stated Akademisk avhandling som med tillstånd av Kungliga Tekniska Högskolan i Stockholm framlägges till offentlig granskning för avläggande av teknologie licentiatexamen i bioteknologi onsdagen den 8 juni klockan 14.00 i Alfa 2, Science for Life Laboratory, Tomtebodavägen 23A, Solna. Abstract This thesis describes the use of antibodies and immunofluorescence for subcellular localization of proteins. The key objective is the creation of an open- source atlas with information on the subcellular location of every human protein. Knowledge of the spatial distribution and the precise location of a protein within a cell is important for its functional characterization, and describing the human proteome in terms of compartment proteomes is important to decipher cellular organization and function. Immunofluorescence and confocal microscopy of cultured cells were used for high-resolution detection of proteins on a high-throughput scale. Critical to immunofluorescence results are sample preparation and specific antibodies. Antibody staining of cells requires fixation and permeabilization, both of which can result in loss or redistribution of proteins and masking of epitopes. A high- throughput approach demands a standardized protocol suitable for the majority of proteins across cellular compartments. Paper I presents an evaluation of sample preparation techniques from which such a single fixation and permeabilization protocol was optimized. Paper II describes the results from applying this protocol to 4000 human proteins in three cell lines of different origin. Paper III presents a strategy for application-specific antibody validation. Antibodies are the key reagents in immunofluorescence, but all antibodies have potential for off-target binding and should be validated thoroughly. Antibody performance varies across sample types and applications due to the competition present and the effect of the sample preparation on antigen accessibility. In this paper application-specific validation for immunofluorescence was conducted using colocalization with fluorescently tagged protein in transgenic cell lines. Keywords: Human proteome, Subcellular localization, Organelles, Immunofluorescence, Fixation, Permeabilization, Antibody validation i Sammanfattning Denna avhandling berör användandet av antikroppar och immunofluorescence för lokalisationsbestämning av proteiner. Målet med arbetet har varit uppbyggnaden av en webbaserad atlas över den subcellulära lokalisationen hos alla mänskliga proteiner. Kunskap om ett proteins lokalisation är en viktig aspekt för att utröna proteinets funktion. Därtill är kunskap om proteomet hos enskilda organeller och subcellulära enheter viktigt för att förstå cellens organisation och funktion. Immunofluorescence och konfokalmikroskopi av odlade celler användes för storskalig lokalisationsbestämning av proteiner med hög precision. För att säkerställa bra resultat i immunofluorescence är provpreparering samt specifika antikroppar av högsta betydelse. Infärgning med antikroppar kräver fixering och permeabilisering av cellerna vilket kan resultera i förlust eller omfördelning av proteiner samt maskering av epitop. För en storskalig ansats till lokalisationsbestämning av alla mänskliga proteiner krävs en standardiserad provförberedning lämplig för majoriteten av alla protein i alla subcellulära strukturer. I artikel I utvärderades ett antal olika fixerings och permeabiliseringsalternativ från vilka ett optimalt protokoll valdes. I artikel II användes detta protokoll för 4000 mänskliga proteiner i tre olika cellinjer. Artikel III presenterar en strategi för applikationsspecifik antikroppsvalidering. Antikroppar är nyckelreagens i immunofluorescence, men alla antikroppar har potential för korsreaktivitet och måste valideras noggrant. Antikroppars prestation varierar mellan provtyper och applikationer beroende på förekomsten av konkurrerande proteiner och provprepareringens påverkan på epitopet. I denna artikel användes överlapp med fluorescent taggat protein i transgena cellinjer för applikationsspecifik validering av antikroppar för immunofluorescence. ii List of publications Ι Stadler C, Skogs M, Brismar H, Uhlén M, Lundberg E. A single fixation protocol for proteome-wide immunofluorescence localization studies. J Proteomics 73(6), 1067-78 (2010) doi: 10.1016/j.jprot.2009.10.012 ΙΙ Fagerberg L, Stadler C, Skogs M, Hjelmare M, Jonasson K, Wiking M, Abergh A, Uhlén M, Lundberg E. Mapping the subcellular protein distribution in three human cell lines. J Proteome Res 10(8), 3766-77 (2011) doi: 10.1021/pr200379a ΙΙΙ Skogs M, Stadler C, Schutten R, Hjelmare M, Gnann C, Poser I, Hyman AA, Uhlén,M Lundberg E. An antibody validation scheme for immunofluorescence using gene tagging. Manuscript Respondents contributions to the included publications Ι Intellectual input in study design and manuscript writing ΙΙ Performed part of the IF experiments ΙΙΙ Majority of study design and experimental planning. Participated in the laboratory work. All analysis. Majority of manuscript writing. iii Additional publications not included in this thesis Fagerberg L, Hallström BM, Oksvold P, Kampf C, Djureinovic D, Odeberg J, Habuka M, Tahmasebpoor S, Danielsson A, Edlund K, Asplund A, Sjöstedt E, Lundberg E, Szigyarto CA, Skogs M, Takanen JO, Berling H, Tegel H, Mulder J, Nilsson P, Schwenk JM, Lindskog C, Danielsson F, Mardinoglu A, Sivertsson A, von Feilitzen K, Forsberg M, Zwahlen M, Olsson I, Navani S, Huss M, Nielsen J, Ponten F, Uhlén M. Analysis of the human tissue-specific expression by genome- wide integration of transcriptomics and antibody-based proteomics. Mol Cell Proteomics 13(2), 397-406 (2014) Danielsson F, Skogs M, Huss M, Rexhepaj E, O'Hurley G, Klevebring D, Pontén F, Gad AK, Uhlén M, Lundberg E. Majority of differentially expressed genes are down-regulated during malignant transformation in a four-stage model. Proc Natl AcaD Sci U S A 110(17), 6853-8 (2013) Fagerberg L, Oksvold P, Skogs M, Algenäs C, Lundberg E, Pontén F, Sivertsson A, Odeberg J, Klevebring D, Kampf C, Asplund A, Sjöstedt E, Al-Khalili Szigyarto C, Edqvist PH, Olsson I, Rydberg U, Hudson P, Ottosson Takanen J, Berling H, Björling L, Tegel H, Rockberg J, Nilsson P, Navani S, Jirström K, Mulder J, Schwenk JM, Zwahlen M, Hober S, Forsberg M, von Feilitzen K, Uhlén M. Contribution of antibody-based protein profiling to the human Chromosome- centric Proteome Project (C-HPP). J Proteome Res 12(6), 2439-48 (2013) Danielsson F, Wiking M, Mahdessian D, Skogs M, Ait Blal H, Hjelmare M, Stadler C, Uhlén M, Lundberg E. RNA deep sequencing as a tool for selection of cell lines for systematic subcellular localization of all human proteins. J Proteome Res 12(1), 299-307 (2013) Jakobsen L, Vanselow K, Skogs M, Toyoda Y, Lundberg E, Poser I, Falkenby LG, Bennetzen M, Westendorf J, Nigg EA, Uhlen M, Hyman AA, Andersen JS. Novel asymmetrically localizing components of human centrosomes identified by complementary proteomics methods. EMBO J 30(8), 1520-35 (2011) iv Abbreviations BAC Bacterial artificial chromosome CDR Complementarity determining region DAPI 4’,6-diamidino-2-phenylindole DNA Deoxyribonucleic acid Fab Fragment antigen binding Fc Fragment crystallizable FPKM Fragments per kilobase of transcript per million mapped reads GFP Green fluorescent protein HPA Human protein atlas IF Immunofluorescence IHC Immunohistochemistry MS Mass spectrometry PFA Paraformaldehyde PTM Posttranslational modification RNA Ribonucleic acid WB Western blot v vi Contents Abstract i List of publications iii Abbreviations v Introduction 1 1. Cell Biology 3 The first description of the small room 4 Cell organization 4 Cell cultivation 5 Visualizing cells 6 2. Genes and proteins 7 Genes 7 Proteins 8 How many genes and proteins do we have? 8 Proteomics 9 Proteomics for subcellular localization 10 3. Antibodies 13 Antibodies in the immune system 13 Antibody structure 14 Antibody diversity 16 Antibodies as research tools 17 Antibody binding 18 Antibody applications 20 Antibody validation 22 Antibody generation projects 23 4. Immunofluorescence 25 Sample preparation 25 Fluorescence microscopy 27 Validation of IF results 28 5. Human Protein Atlas 29 HPA antibodies 30 Subcellular Protein Atlas 30 Validation strategies in the Subcellular Protein Atlas 32 6. Present investigation 35 Paper Ι 35 Paper ΙΙ 36 Paper ΙΙΙ 37 Future perspectives 39 Acknowledgements 41 7. References 43 8. Appendix: Included publications 53 Introduction In the quest to understand the human body we can choose to study it on different levels; organism level, organ level, tissue level, cell level or molecular level. This thesis describes the study of individual cells in the laboratory, which are used as representatives of the cells in the body. Just like the tissues and organs carry out specialized functions in the body, cells are made up of organelles and specialized cellular compartments with distinct functions. The papers included in this thesis were conducted with the aim to determine the constituents
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