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Characterization of the Coelomic Fluid of the Starfish Marthasterias Glacialis in a Wound-Healing Phase Biological Engineering

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Characterization of the Coelomic Fluid of the Starfish Marthasterias Glacialis in a Wound-Healing Phase Biological Engineering Characterization of the coelomic fluid of the starfish Marthasterias glacialis in a wound-healing phase Rita de Albano da Silva Laires Thesis to obtain the Master of Science Degree in Biological Engineering Examination Committee Chairperson: Prof. Duarte Miguel de França Teixeira dos Prazeres (DBE) Supervisors: Prof. Gabriel António Amaro Monteiro (DBE) Dra. Ana Maria de Jesus Bispo Varela Coelho (ITQB) Member of the Committee: Prof. Miguel Nobre Parreira Cacho Teixeira (DBE) November 2012 ii ACKNOWLOGMENTS Todo o trabalho experimental descrito nesta dissertação foi realizado no Laboratório de Espectrometria de Massa do Instituto de Tecnologia Química e Biológica (ITQB), em Oeiras. À minha orientadora, Professora Doutora Ana Varela Coelho, agradeço a oportunidade de ter realizado este trabalho e por ter partilhado comigo os seus conhecimentos científicos. Agradeço ainda a oportunidade de ter ido ao “The Sven Lovén Centre for Marine Sciences” (University of Gothenburg), na Suécia, ao abrigo do projecto ASSEMBLE (Association of European Marine Biological Laboratories) para aprofundar os meus conhecimentos relativos às estrelas-do-mar. Ao meu orientador no IST, Prof. Gabriel Monteiro, agradeço a sua disponibilidade ao longo deste semestre. Estre trabalho não teria sido possível realizar sem a Doutora Kamila Kocí que tanto me ajudou na realização do trabalho desenvolvido no Laboratório. Obrigada pela tua paciência nos momentos de desespero e por tanto me teres ensinado…Obrigada Kami, tenho a certeza que os nossos caminhos se voltarão a cruzar . À Doutora Catarina Franco, agradeço por ter acreditado e apostado em mim. Acredita que também tiveste um papel importante na realização deste trabalho . À Dra. Elisabete Pires, agradeço-te a ajuda fundamental que deste ao longo da minha passagem pelo Laboratório. Sabes bem que foste responsável pelo ponto de viragem deste trabalho . Obrigada Beta… À Doutora Renata Soares, agradeço a partilha dos seus conhecimentos científicos relativos à espectrometria de massa. Obrigada por achares que os meus resultados eram “super interessantes” . Aos meus colegas do Laboratório: Joana, Luís e Miguel, obrigada pelos momentos de boa disposição e pelo bom ambiente que vivemos no Laboratório. Um agradecimento especial também à São, por ter sempre uma palavra amável para mim. Agradeço ainda à minha família, em particular ao meu irmão Miguel que, tal como eu, está a terminar o seu percurso académico e acompanhou-me na escrita desta dissertação. Tenho a certeza que vais conseguir terminar com sucesso. A todos os meus amigos, em especial, Gui, Martine, Ângela, Gonçalo e Irene, obrigada pelo carinho demonstrado por mim desde sempre. A ti, Verde, agradeço-te o apoio incondicional e por me dares força para eu continuar… ii iii ABSTRACT Regenerative potential is expressed to a maximum extent in echinoderms. Starfishes are well known echinoderms, which are capable of reconstructing external appendages and internal organs often subjected to amputation, self-induced or traumatic, rapidly followed by complete successful re- growth of the lost parts. The coelomic fluid that bathes the internal organs of starfish is rich in molecules involved in cell signaling processes. It was intended to study which of these molecules present in the coelomic fluid (bioactive peptides or other low molecular mass compounds) could play an important role in the regeneration process of the starfish Marthasterias glacialis. For that, a mass spectrometry based approach was used to characterize the molecules present in the coelomic fluid of the starfish in the first phase of its regeneration process, a wound-healing phase (48 hours post-arm tip ablation). None of the spectra obtained from MALDI-TOF/TOF MS and ESI-MS showed a fragmentation pattern characteristic for peptides. Instead, asterosaponins were found in high concentration levels in the coelomic fluid of regenerating animals. Asterosaponins are one of the bioactive secondary metabolites from starfish with known immunological, physiological and pharmacological activities, and thus these compounds seem to be promising signaling molecules to study the starfish regeneration process. Keywords: regeneration, starfish, coelomic fluid, mass spectrometry, asterosaponins iv RESUMO O potencial de regeneração tem nos equinodermes a sua máxima expressão. As estrelas-do- mar são equinodermes bem estudados, sendo capazes de reconstruir partes externas do seu corpo e os seus órgãos internos, frequentemente sujeitos a amputação, auto-induzida ou traumática, rapidamente seguido do seu crescimento completo. O fluido celómico que banha todo o interior da estrela-do-mar é rico em moléculas que estão envolvidas em processos de sinalização celular. No presente trabalho, pretendeu-se estudar quais destas moléculas presentes no fluido celómico (péptidos bioactivos ou outros compostos de baixa massa molecular) poderiam desempenhar um papel fundamental no processo de regeneração da estrela-do-mar Marthasterias glacialis. Para tal, foi usada uma abordagem de espectrometria de massa para caracterizar as moléculas presentes no fluido celómico da estrela-do-mar numa primeira fase do processo de regeneração, a fase de cicatrização (48 horas depois da amputação da extremidade de um braço). Nenhum dos espectros MALDI-TOF/TOF MS e ESI-MS obtidos apresentaram padrões de fragmentação típicos de péptidos. Ao invés, foram encontradas asterosaponinas em altas concentrações no fluido celómico de animais em regeneração. As asterosaponinas constituem um dos metabolitos secundários bioactivos das estrelas-do-mar que apresentam conhecidas propriedades imunológicas, fisiológicas e farmacológicas, e como tal, estes compostos aparentam ser moléculas relevantes para o estudo do processo de regeneração de estrelas-do-mar. Palavras-chave: regeneração, estrela-do-mar, fluido celómico, espectrometria de massa, asterosaponinas v TABLE OF CONTENTS ACKNOWLOGMENTS ............................................................................................................................. ii ABSTRACT ............................................................................................................................................. iv RESUMO ..................................................................................................................................................v TABLE OF CONTENTS .......................................................................................................................... vi LIST OF FIGURES ................................................................................................................................ viii LIST OF TABLES .....................................................................................................................................x LIST OF ABREVIATIONS ...................................................................................................................... xii 1. GENERAL INTRODUCTION ........................................................................................................... 1 1.1 Echinoderms ............................................................................................................................ 1 1.2 The animal model: Marthasterias glacialis .............................................................................. 4 1.3 Regeneration in echinoderms.................................................................................................. 5 2. AIM OF THE STUDY ....................................................................................................................... 7 3. AN INTRODUCTION TO MASS SPECTROMETRY ....................................................................... 8 3.1 Electrospray Ionization & Ion-Trap Mass Analyser ............................................................... 10 3.2 Matrix-Assisted Laser Desorption/Ionization & Time-Of-Flight ............................................. 13 3.3 Liquid Chromatography-Mass Spectrometry (LC-MS) .......................................................... 15 4. PRELIMINARY EXPERIMENTS ................................................................................................... 17 4.1 Optimization of methods to obtain a low molecular mass fraction ........................................ 18 4.1.1 Extraction methods tested to obtain a low molecular mass fraction ............................. 18 4.1.2 Evaluation of the extraction methods by LC-ESI-MS .................................................... 20 4.1.2.1 Analysis of the non-spiked CFF samples .................................................................. 20 4.1.2.2 Analysis of the spiked blanks and spiked CFF samples ........................................... 21 4.1.3 Evaluation of UF method with 1D electrophoresis ........................................................ 22 4.2 Methods to detect compounds present in the low molecular mass fraction .......................... 23 4.2.1 UF-Micro-flow LC-spotter-MALDI-TOF/TOF MS ........................................................... 23 4.2.1.1 Micro-flow LC-spotter system .................................................................................... 23 4.2.1.2 Analysis of CFF with UF-Micro-flow LC-spotter-MALDI-TOF/TOF MS ..................... 24 4.2.2 UF-SPE-MALDI-TOF/TOF MS ...................................................................................... 25 5. MATERIALS AND METHODS......................................................................................................
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