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
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:36415 |
| Date | 03 December 2019 |
| Creators | Milentyev, Alexander |
| Contributors | Hoflack, Bernard, Hippler, Michael, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | info:eu-repo/grantAgreement/Deutsche Forschungsgemeinschaft/FOR 2038/233120155// Nanopatterned Organic Matrices in Biological Silica Mineralization |
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