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Chemical Proteomics: Mass Spectrometry-Based Label-Free Quantitative Approach in Cell Systems

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Chemical Proteomics: Mass Spectrometry-Based Label-Free Quantitative Approach in Cell Systems Chemical Proteomics: mass spectrometry-based label-free quantitative approach in cell systems Catarina Guimarães Frazão de Faria Thesis to obtain the Master Science Degree in Biological Engineering Supervisors: PhD Alfonsina D’Amato Prof. Maria Matilde Soares Duarte Marques Examination Committee Chairperson: Prof. Arsénio do Carmo Sales Mendes Fialho Supervisor: Prof. Maria Matilde Soares Duarte Marques Member of the Committee: Dra. Alexandra Maria Moita Antunes October 2018 ACKNOWLEDGMENTS First and foremost, I would like to dedicate this work to my grandparents Maria Helena and José Manuel, who unfortunately did not live long enough to accompany my latest journeys yet had always encouraged me to pursue my goals. The second note goes to my parents, for their categorical support, and for their effort to financially make it possible for me to embark on a lifetime experience: the 6-months-Erasmus in Milan that I had long dreamed about, to do the research internship that prospered this thesis. All and all, they are the people that over the years gave all the tools for me to build my character, to enrich my vision on things, and to thrive. I am sincerely thankful for the opportunity of work and the conditions presented at Unità di Ricerca di Analisi Farmaceutica e Biofarmaceutica, Dipartimento di Scienze Farmaceutiche (DISFARM), Università degli Studi di Milano, by the hands of Prof. Marina Carini and Prof. Giancarlo Aldini, who accepted to receive me, and Alfonsina D’Amato PhD, in the quality of my supervisor. To my Italian colleagues, with whom I shared most of my time at the lab, words cannot describe how incredibly fortunate I feel to have found myself in such a joyful, enthusiastic and caring environment. More than simply being co-workers, we pieced together memories that truly marked my experience abroad, for which I am very grateful. And luckily alongside I had the chance to become somewhat fluent in Italian; se non fosse stato per il loro incentivo quotidiano, non sarei mai riuscita a farlo. Ricorderò sempre tutti i momenti divertenti a pausa pranzo, la loro perplessità con il cibo che portavo, le mille volte che ho detto che non volevo caffè; le serate di beach volley e in palestra, e tutti gli altri programmi che abbiamo fatto fuori dal laboratorio, dagli aperitivi all’Union Club (che odiavo) e pranzi al Doge alla cena portoghese a casa mia, non dimenticando mai le giornate ai laghi di Como e d'Iseo, e in particolare il bellissimo giorno del mio compleanno. E per tutto questo e molto altro, per tutti i ricordi che ho nel cuore, lascio loro una parola di ringraziamento: a Giacomo, dal primo giorno il mio compagno di lavoro in questo progetto; a Laura, Marco, Giovanna, Martina, Ettore, Paola, Nicolas, Federica; e ad Alessandra. I would like as well to thank my internal supervisor at Instituto Superior Técnico, Prof. Matilde Marques, for the guidance and help provided. On another personal note, I would like to give a special thank you to all the friends that supported me throughout the years, particularly during this Erasmus period, which if it had to be summarized in one word it would be dramatic. To my dearest friends from Portugal, some that came to visit, some that were abroad too and I went to visit, and some that I met in Milan; and to my international Erasmus buddies Theresa, Sofija and Tomasz. They say Erasmus is about the people you encounter and those whom you become close with, and it could not be any truer. I am tremendously grateful for the 6 months I got to spend in Milan developing this work; for all the new friendships, the travelling, the chance to expand my horizons and, above all, for allowing me to grow as a person and to acquire life lessons that I will forever recall. 3 PREFACE The work presented in this thesis was performed at Unità di Ricerca di Analisi Farmaceutica e Biofarmaceutica, Dipartimento di Scienze Farmaceutiche (DISFARM), Università degli Studi di Milano, address Via Luigi Mangiagalli 25-20133, Milan, Italy, under the supervision of Prof. Marina Carini and Alfonsina D’Amato PhD, during the period February – July 2018 within the frame of the Erasmus+ programme. It was co-supervised at Instituto Superior Técnico by Prof. Matilde Marques. 4 ABSTRACT For the present project, a novel mass spectrometry-based label-free untargeted method was followed under the paradigm of Quantitative Proteomics. The work developed provides an example of a modern-era explorative proteomic approach in cell systems, supporting the current multidisciplinary context of Chemical Proteomics as a fundamental contributor to drug development. The method was based on applying cellular shotgun proteomics without labelling, making use of the high- resolution OrbitrapTM FusionTM TribridTM mass spectrometer for a technologically advanced mass spectrometric analysis coupled with on-line nano-high performance liquid chromatographic separation, which allows an in- depth identification and characterization of entire proteomes. In this case, it was studied the proteolytic digested proteome of Caco-2 cells upon treatment with 4-hydroxy- 2-nonenal (HNE) in concentration range from 0 to 100 µM. HNE is an electrophile molecule derived from lipid peroxidation of poly-unsaturated fatty acids, known to play a key cytoprotective role in oxidative stress prior to exerting toxic effects. Thus, the aim was to attain an integrative protein profiling of this complex biological matrix to assess protein changes and post-translational modifications that could be related to the beneficial character of HNE in oxidative stress, and the cellular pathways involved in the antioxidant response. From a total of 3354 proteins identified with high-confidence, the ones differentially expressed against the control were brought to closer attention, using various bioinformatic analysis tools to generate protein networks clustered on the premise of relevant biological processes modulated by HNE. These particularly included response to stimulus and stress, detoxification of reactive oxygen species, oxidation metabolism, protein regulation and degradation, nucleosome assembly, inflammation and immune response. As result of the functional proteomic analysis, several protein targets and pathways identified might be further explored and validated for biomarkers of oxidative stress and endorse the design of new bioactive compounds for the treatment of oxidative stress-related conditions such as cancer, neurodegenerative, autoimmune and cardiovascular diseases. In conclusion, a progressive high-tech label-free proteomic approach with human cells is here reported highlighting the search for novel potential beneficial effects of HNE at molecular level, emphasizing the importance of mass spectrometry-based proteomics in pre-clinical pharmaceutical research. Keywords quantitative proteomics, mass spectrometry, functional proteomic analysis, HNE, oxidative stress, antioxidant response 5 Resumo No presente projecto, sob o paradigma da Proteómica Quantitativa, foi seguido um método original de espectrometria de massa sem marcação e não-direccionado. O trabalho desenvolvido fornece um exemplo de uma abordagem moderna de proteómica explorativa em sistemas celulares, no actual contexto multidisciplinar da Proteómica Química como contribuinte fundamental para o desenvolvimento de fármacos. O método baseou-se na aplicação de proteómica celular do tipo shotgun, usando o espectrómetro de massa de alta-resolução OrbitrapTM FusionTM TribridTM para uma análise espectrométrica tecnologicamente avançada acoplada on-line com separação cromatográfica líquida de nano-alto desempenho, que permite a identificação e caracterização aprofundada de proteomas completos. Neste caso, estudou-se o proteoma digerido de células Caco-2 após tratamento com 4-hidroxi-2-nonenal (HNE) em gama de concentração 0 a 100 µM. HNE é uma molécula eletrofílica derivada da peroxidação lipídica de ácidos gordos poli-insaturados, que desempenha um importante papel citprotector durante o stress oxidativo antes de exercer efeitos nocivos. O objectivo passou assim por obter um perfil proteico integrativo desta matriz biológica complexa de modo a avaliar alterações proteicas e modificações pós-tradução que possam relacionar- se com o carácter benéfico do HNE no stress oxidativo e as vias celulares envolvidas na resposta antioxidante. De um total de 3354 proteínas identificadas com nível alto de confiança, aquelas que se mostraram diferencialmente expressas em comparação com os controlos foram analisadas com maior atenção, recorrendo a diversas ferramentas de análise bioinformática para gerar redes de proteínas agrupadas na premissa de processos biológicos relevantes modulados pelo HNE. Estes incluíram particularmente resposta a estímulos e stress, destoxificação de espécies oxigenadas reactivas, metabolismo de oxidação, regulação e degradação proteica, organização de nucleossomas, resposta inflamatória e imunitária. Como resultado de uma análise proteómica funcional, várias proteínas-alvo e vias de interesse identificadas poderão ser exploradas como biomarcadores de stress oxidativo e endossar o desenho de novos compostos bioactivos para o tratamento de doenças relacionadas com o stress oxidativo, por exemplo, cancro e doenças neurodegenerativas, autoimunes e cardiovasculares. Em conclusão, reporta-se aqui uma abordagem proteómica sem marcação com células humanas, progressiva e de alta tecnologia, destacando a procura de novos potenciais efeitos benéficos do HNE ao nível molecular, que enfatiza a importância da proteómica
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