Proteomanalyse von Pflanzen

Titelseite Inhaltsverzeichnis 1. Einleitung 1 2\. Methoden 13 3\. Ergebnisse 46 4\. Diskussion 99 5\. Zusammenfassung 121 6\. Summary 122 7\. Abkuerzungen 123 8\. Literaturverzeichnis 124 9\. Eigene Veroeffentlichungen 138 10\. Danksagung 140Within the course of this PhD thesis twodimensional gelelectrophoresis and MALDI-TOF-MS based methods have been evaluated and developed to fulfil the aim of large scale plant proteomics. Particular attention was paid to the optimisation of proteinextraction, proteinseparation and proteinstaining. In this context a new fractionation based proteinextraction method which gave rise to an 300 % increased display of proteinspots on 2-DE gels could be established. On the basis of the developed techniques we were able to establish a set of 2-DE standardpatterns from 8 different Arabidopsis thaliana tissues. Apart from the optimisation of 2-DE techniques an improved, robust and automated MALDI sample preparation system could be established. The set up of these methods allows the analysis and handling of more than 1000 proteinspots from 2-DE gels per day. In a following step the combination of the 2-DE-and the MALDI protocols will be employed for the large scale identification of a vast portion of the Arabidopsis thaliana proteome. As a primary step it was possible in a set of proof of principle experiments to identify 681 proteinspots from two different Arabidopsis thaliana leaf fraction 2-DE gels. Further we identified 352 proteinspots from an Arabidopsis thaliana silique 2-DE gel. In total the number of these preliminary identified proteins exceeds by far the number of previous published 2-DE proteomic data from Arabidopsis thaliana. In a second proof of principle experiment it was possible to show the increased separation capabilities of 2-DE gels. In this example the identification of differentially expressed proteins from water starved cucumber plants was achieved. This experiment clearly showed the need of two-dimensional separation of proteins from complex mixtures to display differentially expressed proteins, since one-dimensional protein separation is not sufficient to fulfil this task. In a last example the combination of tissue prefractionation techniques with 2-DE and MALDI-MS was used to identify an Arabidopsis thaliana subproteome. In this case the purification of cytosolic 80S ribosomal proteins was achieved by sucrose gradient density centrifugation of Arabidopsis thaliana leaf tissue. In this experiment it was possible to identify a large part (70 %) of the expected protein components of plants cytosolic ribosome from a single gel in a single round of identification. In total a number of 224 proteinspots could be identified from this ribosomal sample.Im Rahmen der vorliegenden Arbeit wurden Techniken ebenso wie Methoden evaluiert und entwickelt, die basierend auf der Verwendung von zweidimensionaler Gelelektrophorese und MALDI-TOF-MS Proteomanalysen von pflanzlichen Geweben ermöglichen. Hierbei wurden zunächst optimierte Methoden für die Proteinextraktion, Proteintrennung und die Proteinfärbung für die 2-DE erarbeitet. So konnte z. B. eine neue fraktionierte Gesamtproteinextraktionsmethode für Pflanzengewebe entwickelt werden, die eine bis zu 300 % verbesserte Ausbeute an Proteinspots auf 2-DE-Gelen im Vergleich zu Standardextraktionsmethoden zulässt. Diese optimierten Methoden wurden in einem weiterführenden Schritt für die Herstellung von 2-DE-Standardmustern aus 8 unterschiedlichen Arabidopsis thaliana-Geweben eingesetzt. Neben der Methodenentwicklung im 2-DE-Bereich wurden im Bereich der MALDI- Probenpräparation und der MALDI-Analyse stabile und automatisierte Protokolle für die Identifikation von 2-DE-Gel-getrennten Proteinen etabliert. Diese Protokolle ermöglichen es, mehr als 1000 Proteinspots pro Tag massenspektrometrisch zu analysieren. Um die reelle Anwendbarkeit und Tauglichkeit der entwickelten Methoden zu dokumentieren, wurden anhand dreier charakteristischer pflanzenspezifischer Fragestellungen Beispielexperimente durchgeführt. So ließen sich in einem ersten Ansatz 681 Proteinspots aus 2 unterschiedlichen Arabidopsis thaliana-Blattproteinextrakten und 352 Proteinspots aus einem Arabidopsis thaliana-Schotenproteinextrakt identifizieren. Die hierbei identifizierten Proteine, die aus nur 3 2-DE-Gelen stammten, übersteigen in ihrer Summe bereits alle bis dato publizierten Daten im Bereich der Arabidopsis thaliana-2-DE-basierten Proteomforschung. In einem zweiten Anwendungsexperiment wurden differentiell regulierte Phloemproteine aus trockengestressten Gurkenpflanzen in 2-DE-Gelen dargestellt und identifiziert. Hierbei zeigte sich, dass diese Art der differentiellen Analyse komplexerer Proteinmischungen das Auflösungsvermögen von herkömmlichen eindimensionalen Trennsystemen übersteigt und somit fast ausschließlich mittels zweidimensionalen Trennsystemen durchgeführt werden sollte. In einem letzten Anwendungsbeispiel, dessen Fokus auf der Analyse des cytosolischen 80S Ribosoms aus Arabidopsis thaliana-Blättern lag, konnte durch die Kombination einer Gewebsvorfraktionierung und der 2-DE ein Großteil (70 %) der bekannten Komponenten des cytosolischen Ribosoms bestimmt werden. Insgesamt ließen sich hierbei 224 Proteinspots aus einem einzigen 2-DE-Gel identifizieren

Analysis of xylem sap proteins from Brassica napus

BACKGROUND: Substance transport in higher land plants is mediated by vascular bundles, consisting of phloem and xylem strands that interconnect all plant organs. While the phloem mainly allocates photoassimilates, the role of the xylem is the transport of water and inorganic nutrients from roots to all aerial plant parts. Only recently it was noticed that in addition to mineral salts, xylem sap contains organic nutrients and even proteins. Although these proteins might have important impact on the performance of above-ground organs, only a few of them have been identified so far and their physiological functions are still unclear. RESULTS: We used root-pressure xylem exudate, collected from cut Brassica napus stems, to extract total proteins. These protein preparations were then separated by high-resolution two-dimensional gel electrophoresis (2-DE). After individual tryptic digests of the most abundant coomassie-stained protein spots, partial peptide sequence information was deduced from tandem mass spectrometric (MS/MS) fragmentation spectra and subsequently used for protein identifications by database searches. This approach resulted in the identification of 69 proteins. These identifications include different proteins potentially involved in defence-related reactions and cell wall metabolism. CONCLUSION: This study provides a comprehensive overview of the most abundant proteins present in xylem sap of Brassica napus. A number of 69 proteins could be identified from which many previously were not known to be localized to this compartment in any other plant species. Since Brassica napus, a close relative of the fully sequenced model plant Arabidopsis thaliana, was used as the experimental system, our results provide a large number of candidate proteins for directed molecular and biochemical analyses of the physiological functions of the xylem under different environmental and developmental conditions. This approach will allow exploiting many of the already established functional genomic resources, like i.e. the large mutant collections, that are available for Arabidopsis

Ultra Performance Liquid Chromatography and High Resolution Mass Spectrometry for the Analysis of Plant Lipids

Holistic analysis of lipids is becoming increasingly popular in the life sciences. Recently, several interesting, mass spectrometry-based studies have been conducted, especially in plant biology. However, while great advancements have been made we are still far from detecting all the lipids species in an organism. In this study we developed an ultra performance liquid chromatography-based method using a high resolution, accurate mass, mass spectrometer for the comprehensive profiling of more than 260 polar and non-polar Arabidopsis thaliana leaf lipids. The method is fully compatible to the commonly used lipid extraction protocols and provides a viable alternative to the commonly used direct infusion-based shotgun lipidomics approaches. The whole process is described in detail and compared to alternative lipidomic approaches. Next to the developed method we also introduce an in-house developed database search software (GoBioSpace), which allows one to perform targeted or un-targeted lipidomic and metabolomic analysis on mass spectrometric data of every kind

Rapid Affinity Purification of Tagged Plant Mitochondria (Mito-AP) for Metabolome and Proteome Analyses

The isolation of organelles facilitates the focused analysis of subcellular protein and metabolite pools. Here we present a technique for the affinity purification of plant mitochondria (Mito-AP). The stable ectopic expression of a mitochondrial outer membrane protein fused to a GFP:Strep tag in Arabidopsis (Arabidopsis thaliana) exclusively decorates mitochondria, enabling their selective affinity purification using magnetic beads coated with Strep-Tactin. With Mito-AP, intact mitochondria from 0.5 g plant material were highly enriched in 30–60 min, considerably faster than with conventional gradient centrifugation. Combining gradient centrifugation and Mito-AP techniques resulted in high purity of >90% mitochondrial proteins in the lysate. Mito-AP supports mitochondrial proteome analysis by shotgun proteomics. The relative abundances of proteins from distinct mitochondrial isolation methods were correlated. A cluster of 619 proteins was consistently enriched by all methods. Among these were several proteins that lack subcellular localization data or that are currently assigned to other compartments. Mito-AP is also compatible with mitochondrial metabolome analysis by triple-quadrupole and orbitrap mass spectrometry. Mito-AP preparations showed a strong enrichment with typical mitochondrial lipids like cardiolipins and demonstrated the presence of several ubiquinones in Arabidopsis mitochondria. Affinity purification of organelles is a powerful tool for reaching higher spatial and temporal resolution for the analysis of metabolomic and proteomic dynamics within subcellular compartments. Mito-AP is small scale, rapid, economic, and potentially applicable to any organelle or to organelle subpopulations

Analysis of the Compartmentalized Metabolome – A Validation of the Non-Aqueous Fractionation Technique

With the development of high-throughput metabolic technologies, a plethora of primary and secondary compounds have been detected in the plant cell. However, there are still major gaps in our understanding of the plant metabolome. This is especially true with regards to the compartmental localization of these identified metabolites. Non-aqueous fractionation (NAF) is a powerful technique for the determination of subcellular metabolite distributions in eukaryotic cells, and it has become the method of choice to analyze the distribution of a large number of metabolites concurrently. However, the NAF technique produces a continuous gradient of metabolite distributions, not discrete assignments. Resolution of these distributions requires computational analyses based on marker molecules to resolve compartmental localizations. In this article we focus on expanding the computational analysis of data derived from NAF. Along with an experimental workflow, we describe the critical steps in NAF experiments and how computational approaches can aid in assessing the quality and robustness of the derived data. For this, we have developed and provide a new version (v1.2) of the BestFit command line tool for calculation and evaluation of subcellular metabolite distributions. Furthermore, using both simulated and experimental data we show the influence on estimated subcellular distributions by modulating important parameters, such as the number of fractions taken or which marker molecule is selected. Finally, we discuss caveats and benefits of NAF analysis in the context of the compartmentalized metabolome

Unraveling local tissue changes within severely injured skeletal muscles in response to MSC-based intervention using MALDI Imaging mass spectrometry

Pre-clinical and clinical studies are now beginning to demonstrate the high potential of cell therapies in enhancing muscle regeneration. We previously demonstrated functional benefit after the transplantation of autologous bone marrow mesenchymal stromal cells (MSC-TX) into a severe muscle crush trauma model. Despite our increasing understanding of the molecular and cellular mechanisms underlying MSC's regenerative function, little is known about the local molecular alterations and their spatial distribution within the tissue after MSC-TX. Here, we used MALDI imaging mass spectrometry (MALDI-IMS) in combination with multivariate statistical strategies to uncover previously unknown peptide alterations within severely injured skeletal muscles. Our analysis revealed that very early molecular alterations in response to MSC-TX occur largely in the region adjacent to the trauma and only to a small extent in the actual trauma region. Using "bottom up" mass spectrometry, we subsequently identified the proteins corresponding to the differentially expressed peptide intensity distributions in the specific muscle regions and used immunohistochemistry to validate our results. These findings extend our current understanding about the early molecular processes of muscle healing and highlights the critical role of trauma adjacent tissue during the early therapeutic response upon treatment with MSC