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Santosh D. Bhosale D ANNALES UNIVERSITATIS TURKUENSIS ANNALES UNIVERSITATIS D 1381 Santosh D. Bhosale D. Santosh SERUM PROTEOMICS TO DETECT EARLY CHANGES IN TYPE 1 DIABETES AND CAROTID ATHEROSCLEROSIS Santosh D. Bhosale Painosalama Oy, Turku , Finland 2018 Turku Painosalama Oy, ISBN 978-951-29-7386-6 (PRINT) ISBN 978-951-29-7387-3 (PDF) TURUN YLIOPISTON JULKAISUJA – ANNALES UNIVERSITATIS TURKUENSIS ISSN 0355-9483 (PRINT) | ISSN 2343-3213 (PDF) Sarja - ser. D osa - tom. 1381 | Medica - Odontologica | Turku 2018 SERUM PROTEOMICS TO DETECT EARLY CHANGES IN TYPE 1 DIABETES AND CAROTID ATHEROSCLEROSIS Santosh D. Bhosale TURUN YLIOPISTON JULKAISUJA – ANNALES UNIVERSITATIS TURKUENSIS Sarja - ser. D osa - tom. 1381 | Medica - Odontologica | Turku 2018 University of Turku Faculty of Medicine Department of Medical Microbiology and Immunology Turku Doctoral Programme of Molecular Medicine (TuDMM) Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland Supervised by Professor Riitta Lahesmaa, M.D., Ph.D. Dr. Robert Moulder, Ph.D. Turku Centre for Biotechnology Turku Centre for Biotechnology University of Turku and Åbo Akademi University of Turku and Åbo Akademi University Turku, Finland University Turku, Finland Reviewed by Adj. Professor Maciej Lalowski, Ph.D. Adj. Professor Tiina Öhman, Ph.D. Meilahti Clinical Proteomics Core Facility Institue of Biochemistry and Helsinki Institue of Life Science (HiLIFE) Helsinki Institue of Life Science (HiLIFE) Faculty of Medicine University of Helsinki, Finland Univerisity of Helsinki, Finland Opponent Professor Ole N. Jensen, Ph.D. Department of Biochemistry and Molecular Biology University of Southern Denmark Odense M, Denmark The originality of this thesis has been checked in accordance with the University of Turku quality assurance system using the Turnitin OriginalityCheck service. Cover image by Santosh D. Bhosale (Created using https://www.wordclouds.com/) ISBN 978-951-29-7386-6 (PRINT) ISBN 978-951-29-7387-3 (PDF) ISSN 0355-9483 (PRINT) ISSN 2343-3213 (PDF) Painosalama Oy – Turku, Finland 2018 To My Family Abstract ABSTRACT Santosh D. Bhosale Serum Proteomics to Detect Early Changes in Type 1 Diabetes and Carotid Ath- erosclerosis University of Turku, Faculty of Medicine, Department of Medical Microbiology and Immunology, Turku Doctoral Programme of Molecular Medicine (TuDMM), Turku Centre for Biotechnology, University of Turku and Åbo Akademi University Turku, Finland, 2018 The detection of early markers is the key issue in predicting the outcome of in- flammatory diseases such as type 1 diabetes and atherosclerosis. Whilst biochem- ical testing approaches have improved prediction of inflammatory diseases, val- idated biomarkers with better diagnostic specificities are still needed. Currently, majority of the disease-related proteomics studies have focused on their end- points. The work presented in this thesis includes the first comprehensive proteomics analyses on serum samples collected from two unique Finnish longi- tudinal cohorts, namely The Diabetes Prediction and Prevention Project (DIPP) and The Cardiovascular Risk in Young Finns Study (YFS), to identify early markers associated with type 1 diabetes and carotid atherosclerosis. Using mass spectrometry (MS)-based quantitative serum proteomics, profiling was carried out to the study temporal variation in pre-diabetic samples and early markers of plaque formation with the T1D and YFS cohorts, respectively. The analyses revealed consistent differences in the abundance of a number of proteins in subjects having an ongoing asymptomatic changes, several of which are func- tionally relevant to the disease process. Taken together, the discovered markers are candidates for further validation studies in an independent cohorts and may be used to characterize an increased risk, progression and early onset of these diseases. Keywords: Serum, Proteomics, iTRAQ, Label-free quantification, T1D, Athero- sclerosis, LC-MS/MS, SRM-MS Tiivistelmä TIIVISTELMÄ Santosh D. Bhosale Tyypin 1 diabeteksen ja ateroskleroosin kehittymiseen liittyvät varhaiset muutok- set seerumiproteomissa Turun yliopisto, Lääketieteellinen tiedekunta, Lääketieteellinen mikrobiologia ja immunologia, Molekyylilääketieteen tohtoriohjelma, Turun biotekniikan keskus, Turun yliopisto ja Åbo akademi Annales Universitatis Turkuensis, Suomi, 2018 Yksi keskeinen haaste tulehduksellisten sairauksien, kuten tyypin 1 diabeteksen ja ateroskleroosin, ennustamisessa on varhaisten tautimarkkerien löytäminen. Vaikka erilaiset biokemialliset testit ovat jo parantaneet tulehdusperäisten sairauksien en- nustamista, uusia tarkempia biomarkkereita tarvitaan edelleen. Tästä huolimatta mo- nissa näiden alojen proteomiikkatöissä on nykyisin keskitytty sairastumishetken tut- kimiseen. Tämän väitöskirjatyön aikana olemme tehneet laajamittaiset prote- omiikka-analyysit seeruminäytteille, jotka on kerätty osana kahta ainutlaatuista suo- malaista seurantatutkimusta: DIPP-tutkimusta (tyypin 1 diabeteksen ennustaminen ja ennaltaehkäisy) ja YFS-tutkimusta (sydän- ja verisuonitautien riski nuorilla suo- malaisilla). Näissä tutkimuksissa seerumiproteomiikkaa hyödynnettiin ensimmistä kertaa varhaisten tyypin 1 diabetes- ja ateroskleroosimarkkerien etsimiseen. Tutkimme tyypin 1 diabeteksen kehittymiseen ja ateroskleroottisten plakkien muodostumiseen liittyviä muutoksia seerumin proteomiprofiileissa massaspektro- metriaan perustuvan kvantitatiivisen proteomiikan avulla. Nämä analyysit paljas- tivat johdonmukaisia eroja lukuisissa proteiineissa myöhemmin sairastuneiden oi- reettomien henkilöiden ja terveinä pysyneiden kontrollien välillä. Monet näistä proteiineista saattavat myös liittyä olennaisesti tautien kehittymiseen. Tutkimuk- sissamme löydetyt markkerit tarjoavat lähtökohdan tuleville validointitutkimuk- sille, ja niitä voitaisiin tulevaisuudessa käyttää yksilön kohonneen sairastumisris- kin, taudin etenemisen sekä taudin varhaisen puhkeamisen kartoittamiseen. Avainsanat: Seerumi, proteomiikka, iTRAQ, Label-free kvantitointi, tyypin 1 diabetes, ateroskleroosi, LC-MS/MS, SRM-MS Table of contents TABLE OF CONTENTS ABSTRACT ........................................................................................................... 4 TIIVISTELMÄ ....................................................................................................... 5 ABBREVIATIONS ................................................................................................ 9 LIST OF ORIGINAL PUBLICATIONS ............................................................. 12 1 INTRODUCTION ....................................................................................... 13 2 REVIEW OF LITERATURE ...................................................................... 15 2.1 Introduction to proteomics: ................................................................. 15 2.2 Proteomics sample preparation: .......................................................... 15 2.3 Separation of proteins and peptides: ................................................... 16 2.3.1 Gel-based methods: ................................................................. 16 2.3.2 Chromatography-based methods ............................................. 17 2.4 Mass spectrometry in proteomics ....................................................... 18 2.4.1 Ionization methods: ................................................................. 18 2.4.2 Different types of mass analyzers: .......................................... 20 2.4.3 Peptide fragmentation: ............................................................ 21 2.4.4 MS-based data acquisition strategies: ..................................... 23 2.4.5 Spectral identifications and database search engines: ............. 27 2.4.6 Quantitative proteomics: ......................................................... 28 2.4.7 Bioinformatics and data analysis: ............................................ 33 2.4.8 Quantitative proteomics for plasma/serum biomarker discovery: ................................................................................ 34 2.5 An introduction to type 1 diabetes (T1D): .......................................... 37 2.5.1 Genetic predisposition and the environmental determinants: . 37 2.5.2 Pancreatic pathology: .............................................................. 39 2.5.3 The need to identify novel risk factors and prospective sampling: ................................................................................. 40 2.5.4 Proteomics of T1D: ................................................................. 42 2.6 An introduction to atherosclerosis: ..................................................... 47 2.6.1 The pathophysiology of atherosclerosis: ................................. 47 2.6.2 The need to identify novel risk factors: ................................... 49 2.6.3 Proteomics of atherosclerosis: ................................................. 50 3 AIMS OF THE STUDY .............................................................................. 55 4 MATERIALS AND METHODS ................................................................ 56 4.1 Biobanks/Cohorts: .............................................................................. 56 4.1.1 The Diabetes Prediction and Prevention Project (DIPP): ....... 56 4.1.2 The Cardiovascular Risk in Young Finn Study (YFS): .......... 56 4.2 Methods used in this study: ................................................................ 57 4.2.1 Immunodepletion of high abundant serum proteins:
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