During infections, bacterial microorganisms initiate profound interactions with mammalian host cells. Usually defense mechanisms of the host destroy intruding bacteria in rapid manner. However, many bacterial pathogens have evolved in a way to avoid these mechanisms. By use of effector molecules, which can be small organic molecules or proteins with enzymatic activity, the host is manipulated on a molecular level. Effectors mediating post-translational modifications (PTMs) are employed by many pathogens to influence the biological activity of host proteins. In the presented thesis, two related PTMs are investigated in detail: Adenylylation, the covalent transfer of an adenosine monophosphate group from adenosine triphosphate onto proteins, and phosphocholination, the covalent transfer of a phosphocholine moiety onto proteins. Over the past years, enzymes mediating these modifications have been discovered in several pathogens, especially as a mechanism to influence the signaling of eukaryotic cells by adenylylating or phosphocholinating small GTPases. However, the development of reliable methods for the isolation and identification of adenylylated and phosphocholinated proteins remains a vehement challenge in this field of research. This thesis presents general procedures for the synthesis of peptides carrying adenylylated or phosphocholinated tyrosine, threonine and serine residues. From the resulting peptides, mono-selective polyclonal antibodies against adenylylated tyrosine and threonine have been raised. The antibodies were used as tools for proteomic research to isolate unknown substrates of adenylyl transferases from eukaryotic cells. Mass spectrometric fragmentation techniques have been investigated to ease the identification of adenylylated proteins. Furthermore, this work presents a new strategy to identify adenylylated proteins. Additionally, small effector molecules are involved in the regulation of infection mechanisms. In this work, the small molecule LAI-1 (Legionella autoinducer 1) from the pathogen Legionella pneumophila, the causative agent of the Legionnaire’s disease, was synthesised together with its amino-derivatives. LAI-1 showed are a clear pharmacological effect on the regulation of the life cycle of L. pneumophila, initiating transmissive traits like motility and virulence. Furthermore, LAI-1 was shown to have an effect on eukaryotic cells as well. Directed motility of the eukaryotic cells was significantly reduced and the cytoskeletal architecture was reorganised, probably by interfering with the small GTPase Cdc42.

Essa dissertação foi subdivida em cinco capítulos que, apesar de relacionados entre, podem ser lidos e compreendidos de forma independente. O primeiro capítulo trata de um breve histórico sobre o espessamento primário em caules de monocotiledôneas. Seu principal objetivo é deixar evidente para o leitor a problemática referente tanto à nomenclatura dos tecidos relacionados ao espessamento primário em monocotiledôneas, quanto explicar as hipóteses que embasam essas nomenclaturas. O segundo capítulo teve como principal objetivo evidenciar que, apesar da discordância de muitos autores da atualidade, existe continuidade dos tecidos endoderme e periciclo entre os órgãos no corpo vegetativo de monocotiledôneas, apresentando indícios que apóiam esse pensamento. Já o terceiro capítulo, Caule, demonstra dados que corroboram o pensamento defendido nessa dissertação: endoderme e periciclo são os tecidos responsáveis pelo espessamento do corpo primário do caule de monocotiledôneas. O quarto capítulo, Folha, discute e demonstra a presença de endoderme (entorno da unidade vascular desse órgão) e periciclo (como parte dessa unidade vascular) em folhas de Zingiberaceae e Costaceae. O quinto e último capítulo aborda a importância dos já mencionados tecidos, quais sejam endoderme e periciclo, na formação da raiz primária. / Since ends of 19th century, arised a great diversity of terminologys to name meristematic region responsible for primary thickening in monocotyledons. Despite of this great number of denominations, a few hypothesis and ideas are utilized to base this great complex of terminologys. This historical chapter aims explain the origin from this terminologys and understand correlations between hypothesis basis. At the end of this explanation, you can understand why the defense of presence of endodermal and pericycle tissue as the two meristematic tissues responsible for this primary thickening in monocotyledonous rather than the currently most accepted name of \"PTM\", a single meristem advocated by many authors. Despite of many authors consider fact the continuity between the vascular tissues of stem and leaf and stem and root, few authors consider that the endodermis (between stem and leaf and between stem and root) and pericycle (between stem and leaf and stem and between root) of these organs may be continuous. It was shown, with verification of the continuity of the tissues mentioned above, the plant is a unit and can be found in the root and leaves the same tissues found in the stem, in this case, the main tissues examined in this chapter: endodermis and pericycle. This chapter aims to discuss, demonstrate, and finally prove the continuity of the tissues mentioned in Zingiberaceae, and also describe the morphology of these tissues in each of the aforementioned organs. The stems of monocots has been the subject of studies since the nineteenth century. But despite the large amount of work produced since then, there is still controversy regarding the primary tissue responsible for thickening of this organ. The families Zingiberaceae Costaceae and serve as a model for which more data were collected showing not only the presence of endodermis and pericycle in the stem, but also to show that these are the meristematic tissues responsible for primary thickening in this organ. It was observed in the underground stem, the presence of strips of Caspary in the region farthest from the apex. Already in the air stem, the endodermis was seen only parenchymatous, without any morphological alteration in its walls. The meristematic pericycle was found in the underground stem, but in the aerial stems it is pluriseriated and have cell walls thick. Many authors recognize that the leaf is a projection of stem. But despite this recognition of the origin of this organ, few authors admit that there is continuity between the tissue found in these two organs. This chapter aims to show that tissue found in the stem, which are endodermis and pericycle, are also present in the leaves of species of Zingiberaceae and Costaceae families. Although not possible to observe strips of Caspary involving the vascular unit found in the leaves, could be observed in leaf expansion an accumulation of phenolic substances in tissue, facilitating the visualization of the cells corresponding to the endodermis. The pericycle, forming pericycle fibers was also observed and described. The root is one of the most preserved organs of all vegetative organs of vascular plants. This is the only organ where the tissues endodermis and pericycle are found in any textbook and that are part of primary body of the root. But despite this recognition, the endodermis is not seen by the authors in general, as being important for the formation of the root cortex. This chapter shows the presence of endodermis with meristematic activity, and demonstrate its derivatives (DEMs) in the root cortex of species of families Zingiberacaea and Costaceae.

Cu/Zn Superoxide Dismutase (SOD1), is a ubiquitous antioxidant enzyme with several emerging roles outside of its canonical function. SOD1 is also emerging in central roles in cancer and neurodegenerative pathologies. Little is known about SOD1 regulation, particularly at a post-translational level. Post-translational modifications (PTMs) play an important role in enabling proteins to rapidly respond to their environment. Therefore, identifying specific PTMs involved in protein regulation represents a powerful opportunity to interfere with any associated pathologies. This work employs proteomics to identify mechanisms of post-translation regulation on cell survival signaling proteins. We focused on SOD1, which protects cells from oxidative stress. We found that acylation of K122 on SOD1, while not impacting SOD1 catalytic activity, suppressed the ability of SOD1 to inhibit mitochondrial metabolism at respiratory complex I. We found that deacylase depletion increased K122 acylation on SOD1, which blocked suppression of respiration in a K122-dependent manner. In addition, we found that acyl-mimicking mutations at K122 decreased SOD1 accumulation in mitochondria, initially hinting that SOD1 may inhibit respiration directly within the intermembrane space (IMS). However, surprisingly, we found that forcing the K122 acyl mutants into the mitochondria with an IMS-targeting tag did not recover their ability to suppress respiration. Moreover, we found that suppressing or boosting respiration levels toggled SOD1 in or out of the mitochondria, respectively. These findings place SOD1-mediated inhibition of respiration upstream of its mitochondrial localization. Interestingly, we also found that K122 acyl mutants were sufficient to prevent mitochondrial accumulation of the G93A SOD1 clinical mutant. We observed increased autophagic activity in G93A expressing cells compared to WT or G93A/K122-acyl mimic double mutants, and found that this double mutant was just as prone to aggregate as G93A SOD1—suggesting that SOD1 aggregation is more toxic when in the mitochondria. We observed increased protein turnover rates in cells expressing SOD1 G93A, in support of increased autophagy. Lastly, deletion-rescue experiments show that a respiration-defective mutant of SOD1 is also impaired in its ability to rescue cells from toxicity caused by SOD1 deletion. Together, these data suggest a new interplay between SOD1 acylation, metabolic regulation, SOD1 aggregate toxicity, and SOD1-mediated cell survival.

No description available.

Essa dissertação foi subdivida em cinco capítulos que, apesar de relacionados entre, podem ser lidos e compreendidos de forma independente. O primeiro capítulo trata de um breve histórico sobre o espessamento primário em caules de monocotiledôneas. Seu principal objetivo é deixar evidente para o leitor a problemática referente tanto à nomenclatura dos tecidos relacionados ao espessamento primário em monocotiledôneas, quanto explicar as hipóteses que embasam essas nomenclaturas. O segundo capítulo teve como principal objetivo evidenciar que, apesar da discordância de muitos autores da atualidade, existe continuidade dos tecidos endoderme e periciclo entre os órgãos no corpo vegetativo de monocotiledôneas, apresentando indícios que apóiam esse pensamento. Já o terceiro capítulo, Caule, demonstra dados que corroboram o pensamento defendido nessa dissertação: endoderme e periciclo são os tecidos responsáveis pelo espessamento do corpo primário do caule de monocotiledôneas. O quarto capítulo, Folha, discute e demonstra a presença de endoderme (entorno da unidade vascular desse órgão) e periciclo (como parte dessa unidade vascular) em folhas de Zingiberaceae e Costaceae. O quinto e último capítulo aborda a importância dos já mencionados tecidos, quais sejam endoderme e periciclo, na formação da raiz primária. / Since ends of 19th century, arised a great diversity of terminologys to name meristematic region responsible for primary thickening in monocotyledons. Despite of this great number of denominations, a few hypothesis and ideas are utilized to base this great complex of terminologys. This historical chapter aims explain the origin from this terminologys and understand correlations between hypothesis basis. At the end of this explanation, you can understand why the defense of presence of endodermal and pericycle tissue as the two meristematic tissues responsible for this primary thickening in monocotyledonous rather than the currently most accepted name of \"PTM\", a single meristem advocated by many authors. Despite of many authors consider fact the continuity between the vascular tissues of stem and leaf and stem and root, few authors consider that the endodermis (between stem and leaf and between stem and root) and pericycle (between stem and leaf and stem and between root) of these organs may be continuous. It was shown, with verification of the continuity of the tissues mentioned above, the plant is a unit and can be found in the root and leaves the same tissues found in the stem, in this case, the main tissues examined in this chapter: endodermis and pericycle. This chapter aims to discuss, demonstrate, and finally prove the continuity of the tissues mentioned in Zingiberaceae, and also describe the morphology of these tissues in each of the aforementioned organs. The stems of monocots has been the subject of studies since the nineteenth century. But despite the large amount of work produced since then, there is still controversy regarding the primary tissue responsible for thickening of this organ. The families Zingiberaceae Costaceae and serve as a model for which more data were collected showing not only the presence of endodermis and pericycle in the stem, but also to show that these are the meristematic tissues responsible for primary thickening in this organ. It was observed in the underground stem, the presence of strips of Caspary in the region farthest from the apex. Already in the air stem, the endodermis was seen only parenchymatous, without any morphological alteration in its walls. The meristematic pericycle was found in the underground stem, but in the aerial stems it is pluriseriated and have cell walls thick. Many authors recognize that the leaf is a projection of stem. But despite this recognition of the origin of this organ, few authors admit that there is continuity between the tissue found in these two organs. This chapter aims to show that tissue found in the stem, which are endodermis and pericycle, are also present in the leaves of species of Zingiberaceae and Costaceae families. Although not possible to observe strips of Caspary involving the vascular unit found in the leaves, could be observed in leaf expansion an accumulation of phenolic substances in tissue, facilitating the visualization of the cells corresponding to the endodermis. The pericycle, forming pericycle fibers was also observed and described. The root is one of the most preserved organs of all vegetative organs of vascular plants. This is the only organ where the tissues endodermis and pericycle are found in any textbook and that are part of primary body of the root. But despite this recognition, the endodermis is not seen by the authors in general, as being important for the formation of the root cortex. This chapter shows the presence of endodermis with meristematic activity, and demonstrate its derivatives (DEMs) in the root cortex of species of families Zingiberacaea and Costaceae.

<p>  </p> <p>Protein methylation is one of the most common protein posttranslational modifications (PTMs), within which protein α-N-terminal methylation is largely underexplored. Protein α-N-terminal methylation has been implicated in disease development, including cancer and neurodegenerative diseases, but the physiological and pathological roles of this PTM is not well understood. Protein α-N-terminal methylation modifies the free α-amino group on the protein N-termini and adds between one and three methyl groups by α-N-terminal methyltransferases. It has been shown that protein α-N-terminal methylation is conserved across prokaryotes and eukaryotes. The identification and characterization of the two α-N-terminal methyltransferases in humans, NTMT1 and NTMT2, and their homolog in yeast, Tae1, shows a high conserved substrate recognition and possible shared biological roles. α-N-terminal methyltransferases in humans and yeast recognize substrates with a canonical N-terminal motif, X1-P2-[K/R]3 (X=A, S, P or G after the initial M is cleaved). However, most of the proteins containing the canonical motif have not been studied and identified as substrates. In this study, we use a yeast as a model to explore the substrate members in the protein α-N-terminal methylome and understand the potential regulatory mechanisms. </p> <p>We characterized the yeast phenotypes associated with a <em>TAE1 </em>deletion strain, including increased resistance to heat stress, oxidative stress and paromomycin, and increased sensitivity to benomyl. We also extended the substrate repertoire by validating the presence of α-N-terminal methylation on six substrates by mass spectrometry. Furthermore, we investigate how α-N-terminal methylation could regulate Hsp31, a multifunctional heat shock protein that is associated with yeast heat response and oxidative response. Results suggest that methylation might regulate the localization of Hsp31, rather than directly regulating Hsp31 chaperone activity or methylglyoxalase activity. Alternatively, we developed another methodology to explore the α-N-terminal methylome without motif restriction by repurposing public mass spectrometry datasets for α-N-terminal methylation events in both yeast and humans. We found about 1-2 % of the total proteome are α-N-terminally methylated. Interestingly, the majority of the α-N-terminal methylation events were not on the canonical motif sequence. This indicates a more prevalent existence of α-N-terminal methylation.</p>

No description available.

Les modifications de protéines, qu’elles soient post-traductionnelles (PTMs) ou induites chimiquement, ont une influence considérable sur l'activité des protéines. Des méthodes de spectrométrie de masse (MS) HRMS, MS/MS CID et ETD, et de biochimie ont été développées pour la caractérisation structurale et cinétique de complexes protéine-ligand et de PTMs, dans le but de disséquer leur mécanisme et de concevoir des médicaments covalents contre des protéines liant des protéases, des kinases, ou l'ADN. La MS combinée avec des outils biochimiques a permis de séquencer l'inhibiteur de protéases grégline, et de détecter une PTM originale. De même, la transposase MOS1, modèle de l'intégrase du VIH pour la conception d'inhibiteurs, s'avère être à la fois acétylée et phosphorylée. Pour la lyase Abf2, une stratégie de piégeage, purification, protéolyse et hydrolyse ADN du complexe covalent Abf2-ADN, couplée à l’analyse MS, a été développée. Enfin, l’interaction entre le surpresseur de métastase hPEBP1 et la locostatine a été disséquée sur la protéine entière et par approche bottom-up. La locostatine s’hydrolyse en butyrate après fixation. Afin d’identifier le site ciblé par la locostatine, les conditions de réaction et de protéolyse ont été optimisées. La présence de réactions non spécifiques a conduit au développement 1) d'un modèle mathématique permettant de déterminer la fraction de liaison optimale pour discriminer le site spécifique des sites non-spécifiques, et 2) d'une méthode pour la quantification parallèle et exhaustive du degré de modification de tous les sites modifiés d'une protéine. Ces outils sont applicables aux ligands covalents de protéines et/ou à leurs PTMs. / Protein modifications, whether post-translational (PTMs) or chemically induced, play a crucial role on the activity of proteins. Mass spectrometry (MS) techniques such as HRMS, CID/ETD MS/MS, and biochemistrybased methods for structural and kinetic characterization of protein-ligand complexes and PTMs have been developed. MS combined with several biochemical tools has been used to sequence the proteinase inhibitor gregline and to detect a novel PTM. A similar approach shows that the transposase MOS1, a model for the design of HIV integrase inhibitors, is both phosphorylated and acetylated. For the lyase Abf2, a strategy of trapping, purification, proteolysis, and DNA hydrolysis of the Abf2-DNA covalent complex, coupled to MS analysis, has been developed. Finally, the interaction between the metastasis suppressor hPEBP1 and locostatin was dissected. Upon binding to hPEBP1, locostatin undergoes hydrolysis. To identify the site targeted by locostatin, the conditions of reaction and proteolysis were optimized. The qualitative approach reveals the presence of non-specific reactions, leading to the development of 1) a mathematical model to determine the optimum bound fraction for discriminating the specific site from non-specific sites, and 2) a method for the parallel and exhaustive quantification of the degree of modification of all modified sites of a protein. These tools are widely applicable to covalent protein ligands and/or PTMs.

Kieselalgen (Diatomee) sind eukaryotische einzellige Algen die hochspezifische Proteine (sogenannte Silaffine) erzeugen, um ‘nanopatterned’ Silica-Zellwände herzustellen. Diese Proteine zeigen geringe oder gar keine Homologie innerhalb der Diatomeen Gattung und sind ausgiebig (extensiv) posttranslatorisch modifiziert. Zum Unterschied zu konventioneller Modifikation (z.B. Phosphorylierung und Glykosylierung) weisen Lysinreste von Silaffinen einige Polyaminketten mit sehr heterogenen molekularen Strukturen auf. Diese Modifikationen sind spezifisch für Kieselalgen und spielen somit hypothetisch eine Rolle in der Biosilica-Synthese. Allerdings sind Lysin Polyamin Modifikationen, modifizierte Proteine und modifizierte Stellen kaum charakterisiert. Um diese Frage zu beantworten entwickelten wir eine Methode Polyamine zu quantifizieren und die Position von Polyamin-Modifikationen in engverwandte Proteine zu identifizieren (in morphologisch unterschiedliche Diatomeen Thalassiosira pseudonana, T. oceanica und Cyclotella cryptica). Wir zeigten, dass das Gesamtmuster von Polyaminender phylogenetischen Nähe dieser Kieselalgenarten folgt und dass diese Polyaminmodifikationen an Konsensusstellen sogar in Proteinen auftraten, die keine Sequenzähnlichkeit zeigten.:CONTENTS Summary Zusammenfassung List of figures List of tables Abbreviations 1 Introduction 1.1 Diatoms 1.2 Diatom biosilica 1.2.1 Biosilicification in nature 1.2.2 Diatom biosilica structure and cell cycle 1.2.3 The cell biology of biosilica morphogenesis 1.3 The role of polyamine PTMs in diatom biosilicification 1.3.1 Identifying biomolecules associated with diatom biosilica 1.3.2 PTM complexity of biosilica-associated proteins 1.3.3 Lysine ε-polyamine PTMs in biosilica-associated proteins 1.4 Mass spectrometry in PTM discovery 1.4.1 Modification-specific proteomics 1.4.2 Analysis of polyamine-modified lysines by MS 1.4.3 Fractionation of proteins and peptides prior to MS 1.4.4 MS/MS analysis in modification-specific proteomics 1.4.5 Bioinformatics tools for modification-specific proteomics 1.5 Rationale of the thesis 2 Aim of the thesis 3 Results and discussion 3.1 A method for analysis of ε-polyamine PTMs 3.1.1 Establishing a method to analyse ε-polyamines 3.1.2 Method applicability for lysine PTM profiling 3.1.3 Profiling of lysine PTMs in silaffin-3 3.2 Profiling lysine PTMs in biosilica extracts 3.2.1 Lysine PTM profile and characteristic fragments 3.2.2 Elucidation of phosphopolyamine structures 3.2.3 LysinePTMprofilesofAFSMextracts 3.2.4 Comparison of AFIM and AFSM profiles in T. pseudonana 3.2.5 Phylogenetic relationship across three diatom species 3.3 PTM localization and discovery of consensus motifs 3.3.1 Multiple protease strategy for mapping lysine PTMs 3.3.2 Selection of deprotection technique 3.3.3 Mapping lysine PTMs on tpSil3 using iterative search strategy 3.3.4 Deconvolution of raw MS/MS spectra 3.3.5 PTM mapping by polyamine-specific fragments 3.3.6 Identification of consensus motifs harboring lysine PTMs 4 Conclusions and Outlook 5.1 Synthesis of polyamine standards 5.2 Isolation of biosilica-associated proteins 5.3 Expression of tpSil3 from synthetic gene 5.4 HCl hydrolysis 5.5 AQC-derivatization of amino acids and polyamines 5.6 LC-MS/MS analysis of QAC-derivatives 5.7 Amino acid measurement using UV-detection 5.8 Direct infusion MS/MS analysis 5.9 Acetylation of phosphopolyamines 5.10 31P-NMR measurements 5.11 Deglycosylation with TFMS 5.12 Treatment with HF·pyridine soluble complex 5.13 Anhydrous HF-treatment 5.14 Protein analysis by GeLC-MS/MS 5.15 Proteomics data processing A Appendix B Bibliography Acknowledgments Publications Declaration / Erklärung / Diatoms are eukaryotic unicellular algae that employ highly specialized proteins called silaffins for making nanopatterned silica-based cell walls. These proteins share little or no homology across diatom species and are extensively post-translationally modified. Apart from conventional modifications (e. g., phosphorylation and glycosylation) lysine residues of silaffins bear polyamine chains with highly heterogeneous molecular structure. The latter appear to be specific for silicifying organisms and therefore hypothesized to play a key role in biosilica synthesis. However, polyamine modifications of lysines, modified proteins, and modification sites remain poorly characterized. To address these questions, we developed a method to quantify polyamines and identify sites of polyamine modifications in proteins from phylogenetically closely related, yet morphologically distinct diatoms Thalassiosira pseudonana, T. oceanica, and Cyclotella cryptica. We demonstrated that the overall pattern of polyamines followed the phylogenetic proximity across these diatom species and showed that polyamine modifications occurred at consensus sites even in proteins showing no sequence similarity.:CONTENTS Summary Zusammenfassung List of figures List of tables Abbreviations 1 Introduction 1.1 Diatoms 1.2 Diatom biosilica 1.2.1 Biosilicification in nature 1.2.2 Diatom biosilica structure and cell cycle 1.2.3 The cell biology of biosilica morphogenesis 1.3 The role of polyamine PTMs in diatom biosilicification 1.3.1 Identifying biomolecules associated with diatom biosilica 1.3.2 PTM complexity of biosilica-associated proteins 1.3.3 Lysine ε-polyamine PTMs in biosilica-associated proteins 1.4 Mass spectrometry in PTM discovery 1.4.1 Modification-specific proteomics 1.4.2 Analysis of polyamine-modified lysines by MS 1.4.3 Fractionation of proteins and peptides prior to MS 1.4.4 MS/MS analysis in modification-specific proteomics 1.4.5 Bioinformatics tools for modification-specific proteomics 1.5 Rationale of the thesis 2 Aim of the thesis 3 Results and discussion 3.1 A method for analysis of ε-polyamine PTMs 3.1.1 Establishing a method to analyse ε-polyamines 3.1.2 Method applicability for lysine PTM profiling 3.1.3 Profiling of lysine PTMs in silaffin-3 3.2 Profiling lysine PTMs in biosilica extracts 3.2.1 Lysine PTM profile and characteristic fragments 3.2.2 Elucidation of phosphopolyamine structures 3.2.3 LysinePTMprofilesofAFSMextracts 3.2.4 Comparison of AFIM and AFSM profiles in T. pseudonana 3.2.5 Phylogenetic relationship across three diatom species 3.3 PTM localization and discovery of consensus motifs 3.3.1 Multiple protease strategy for mapping lysine PTMs 3.3.2 Selection of deprotection technique 3.3.3 Mapping lysine PTMs on tpSil3 using iterative search strategy 3.3.4 Deconvolution of raw MS/MS spectra 3.3.5 PTM mapping by polyamine-specific fragments 3.3.6 Identification of consensus motifs harboring lysine PTMs 4 Conclusions and Outlook 5.1 Synthesis of polyamine standards 5.2 Isolation of biosilica-associated proteins 5.3 Expression of tpSil3 from synthetic gene 5.4 HCl hydrolysis 5.5 AQC-derivatization of amino acids and polyamines 5.6 LC-MS/MS analysis of QAC-derivatives 5.7 Amino acid measurement using UV-detection 5.8 Direct infusion MS/MS analysis 5.9 Acetylation of phosphopolyamines 5.10 31P-NMR measurements 5.11 Deglycosylation with TFMS 5.12 Treatment with HF·pyridine soluble complex 5.13 Anhydrous HF-treatment 5.14 Protein analysis by GeLC-MS/MS 5.15 Proteomics data processing A Appendix B Bibliography Acknowledgments Publications Declaration / Erklärung

Master of Science / Biochemistry / John Tomich / One of the important keys to characterize the biological function of a protein is the study of post-translational modification (PTM). Formation of disulfide bond linkages between cysteine residues within a protein is a common PTM which not only contributes to folding and stabilizing the protein structure, but also to accomplishing its native function. Therefore, the study and discovery of structural-functional relationships of expressed proteins using an isolated proteomics approach has been one of the biggest advances within the field of structural biology in recent years. In this study, rapid disulfide bond mapping of freshly obtained equine serum albumin (ESA) was performed using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Highly sensitive MALDI-TOF MS is commonly used for the investigation of disulfide bond linkages in the proteomics field. However, it has also been known that the presence of disulfide bond linkages absorbs the energy which is created by the cysteine-cysteine kinetic vibration, resulting in a decrease of the instrumental sensitivity. To overcome this problem, the disulfide bond mapping method was optimized by applying a combination of chemical labeling, proteolytic enzymes, and matrices. With the optimized method, we were also able to achieve high protein sequence coverage. Obtaining higher sequence coverage of a protein provides more information about a protein which helps to identify the protein by peptide mass fingerprint (PMF) technique. These analyses eventually contribute to the estimation of the possible PTM sites.