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High-throughput protein analysis using mass spectrometry-based methods Tove Boström KTH Royal Institute of Technology School of Biotechnology Stockholm 2014 c Tove Boström 2014 KTH Royal Institute of Technology School of Biotechnology Division of Protein Technology AlbaNova University center SE-106 91 Stockholm Sweden Paper I c 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Paper II c 2012 American Chemical Society Paper IV c 2014 American Chemical Society Paper V c 2014 The American Society for Biochemistry and Molecular Biology, Inc. ISBN 978-91-7595-292-5 TRITA-BIO Report 2014:15 ISSN 1654-2312 Printed by Universitetsservice US-AB 2014 "If you can dream it, you can do it." - Walt Disney Abstract In the field of proteomics, proteins are analyzed and quantified in high numbers. Protein analysis is of great importance and can for example generate information regarding protein function and involvement in disease. Different strategies for protein analysis and quan- tification have emerged, suitable for different applications. The focus of this thesis lies on protein identification and quantification using different setups and method development has a central role in all included papers. The presented research can be divided into three parts. Part one describes the develop- ment of two different screening methods for His6-tagged recombinant protein fragments. In the first investigation, proteins were purified using immobilized metal ion affinity chro- matography in a 96-well plate format and in the second investigation this was downscaled to nanoliter-scale using the miniaturized sample preparation platform, integrated selective enrichment target (ISET). The aim of these investigations was to develop methods that could work as an initial screening step in high-throughput protein production projects, such as the Human Protein Atlas (HPA) project, for more efficient protein production and purification. In the second part of the thesis, focus lies on quantitative proteomics. Protein fragments were produced with incorporated heavy isotope-labeled amino acids and used as internal standards in absolute protein quantification mass spectrometry experiments. The aim of this investigation was to compare the protein levels obtained using quanti- tative mass spectrometry to mRNA levels obtained by RNA sequencing. Expression of 32 different proteins was studied in six different cell lines and a clear correlation between protein and mRNA levels was observed when analyzing genes on an individual level. The third part of the thesis involves the antibodies generated within the HPA project. In the first investigation a method for validation of antibodies using protein immunoenrichment coupled to mass spectrometry was described. In a second study, a method was developed where antibodies were used to capture tryptic peptides from a digested cell lysate with spiked in heavy isotope-labeled protein fragments, enabling quantification of 20 proteins in a multiplex format. Taken together, the presented research has expanded the pro- teomics toolbox in terms of available methods for protein analysis and quantification in a high-throughput format. Keywords: Proteomics, mass spectrometry, affinity proteomics, immunoenrichment, immunoprecipitation, IMAC, screening, protein production, protein purification, ISET, quantification, SILAC, stable isotope standard, antibody validation I Sammanfattning I det forskningsområde som kallas proteomik studeras proteiner i stor skala, det vill säga många proteiner parallellt. Genom att studera proteiner kan man till exempel få informa- tion om proteiners funktion och huruvida ett protein är involverat i en sjukdomsprocess. I denna avhandling har proteiner analyserats och kvantifierats på olika sätt och i alla inkluderade artiklar har metodutveckling haft en central roll. Forskningen som presenteras i denna avhandling kan delas in i tre delar. Den första delen beskriver två nya screeningmetoder för His6-taggade rekombinanta proteinfragment. I ett första projekt renades proteinerna med immobiliserad metalljonsaffinitetskromatografi i ett 96-brunnsformat och i ett andra projekt skalades detta ner till nanoliter-format genom att använda provberedningsplattformen ISET. Målet med dessa projekt var att utveckla metoder som skulle kunna användas som ett initialt screeningsteg vid storskalig protein- produktion för att öka effektiviteten. I del två ligger fokus på kvantifiering av proteiner. Proteinfragment producerades med aminosyror med inkorporerade tunga isotoper och an- vändes sedan som interna standarder i absolut kvantifiering med masspektrometri. Målet var här att jämföra proteinnivåer med motsvarande mRNA-nivåer, vilka bestämts med RNA-sekvensering. Koncentrationen av 32 proteiner bestämdes i sex olika cellinjer och det fanns en tydlig korrelation mellan protein och mRNA när generna studerades indi- viduellt. I den sista delen av avhandlingen användes antikroppar som tagits fram inom proteinatlas-projektet (HPA). I ett projekt utvecklades en metod för validering av an- tikroppar genom att först använda antikropparna för anrikning av målproteinerna från ett cellysat och sedan med masspektrometri bestämma proteinernas identitet. I ett andra projekt användes antikropparna istället för att fånga ut peptider från ett trypsinklyvt cellysat. I en setup där tunga isotopinmärkta proteinfragment adderades till lysatet före klyvning kunde 20 proteiner kvantifieras parallellt. Sammantaget har de presenterade projekten bidragit till att möjligheterna för parallell proteinanalys och kvantifiering har ökat. II List of publications This thesis is based on the five publications listed below. The publications are referred to by Roman numbers (I-V) and are included in the Appendix of the thesis. Paper I Tegel H.*, Yderland L.*, Boström T., Eriksson C., Ukko- nen K., Vasala A., Neubauer P., Ottosson J. and Hober S. Parallel production and verification of protein products us- ing a novel high-throughput method. Biotechnol J (2011) vol. 6 (8) pp. 1018-25 Paper II Adler B.*, Boström T.*, Ekström S., Hober S. and Lau- rell T. Miniaturized and automated high-throughput veri- fication of proteins in the ISET platform with MALDI MS. Anal Chem (2012) vol. 84 (20) pp. 8663-9 Paper III Boström T., Danielsson F., Lundberg E., Tegel H., Jo- hansson H. J., Lehtiö J., Uhlén M., Hober S., and Takanen J. O. Investigating the correlation of protein and mRNA levels in human cell lines using quantitative proteomics and transcriptomics. Submitted Paper IV Boström T., Johansson H. J., Lehtiö J., Uhlén M. and Hober S. Investigating the applicability of antibodies gen- erated within the Human Protein Atlas as capture agents in immunoenrichment coupled to mass spectrometry. J Proteome Res (2014) vol. 13 (10) pp. 4424-35 Paper V Edfors F.*, Boström T.*, Forsström B., Zeiler M., Jo- hansson H., Lundberg E., Hober S., Lehtiö J., Mann M. and Uhlén M. Immuno-proteomics using polyclonal anti- bodies and stable isotope labeled affinity-purified recom- binant proteins. Mol Cell Proteomics (2014) vol. 13 (6) pp. 1611-24 *Both authors contributed equally to the work. III Published work not included in the thesis Boström T.*, Nilvebrant J.* and Hober S. Purification systems based on bacterial surface proteins. Protein Purification, R. Ahmad (Ed.), (2012) ISBN: 978-953-307-831-1, InTech *Both authors contributed equally to the work. IV Contributions to the included papers Paper I Performed all experimental work and data analysis to- gether with coauthor. Wrote the manuscript together with coauthors. Paper II Performed all experimental work together with coauthor. Wrote the manuscript together with coauthors. Paper III Performed all experimental work except cell cultivation and performed all MS data analysis. Wrote the manuscript together with coauthors. Paper IV Performed all experimental work and all MS data analysis. Wrote the manuscript together with coauthors. Paper V Performed cell cultivation, MS sample preparation and all MS data analysis. Produced and quantified all heavy isotope-labeled PrESTs. Contributed to the writing of the manuscript. V Abbreviations 2D-GE two-dimensional gel electrophoresis Ab antibody ABD albumin binding domain ABP albumin binding protein ACM antibody colocalization microarray APEX absolute protein expression AQUA absolute quantification AUC area under curve BAC bacterial artificial chromosome BCA bicinchoninic acid cAb capture antibody CDR complementary determining region CIMS context-independent motif specific CR cross reactivity dAb detection antibody DARPin designed ankyrin repeat protein DNA deoxyribonucleic acid DWP deep well plate EGTA ethylene glycol tetraacetic acid EIA enzyme immunoassay ELISA enzyme-linked immunosorbent assay emPAI exponentially modified protein abundance index ESI electrospray ionization EtEP equimolarity through equalizer peptide Fab fragment antigen binding FASP filter-aided sample preparation Fc fragment crystallizable FT-ICR fourier transform ion cyclotron resonance GFP green fluorescent protein GPS global proteome survey His6 hexahistidine HPA human protein atlas IBAQ intensity-based absolute quantification ICAT isotope coded affinity tag IEF isoelectric focusing Ig immunoglobulin IMAC immobilized metal ion affinity chromatography iMALDI immuno-MALDI IRMA immunoradiometric assay ISET integrated selective enrichment target iTRAQ isobaric tags for relative and absolute quantification VI LC liquid chromatography LFQ label free quantification mAb monoclonal antibody MALDI matrix-assisted laser desorption ionization MRM multiple reaction monitoring mRNA messenger
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