Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Dept. of Chemistry & Biomolecular Sciences, 2009. / Bibliography: p. 200-212. / Introduction -- Shotgun proteomic analysis of rat liver membrane proteins -- A combination of immobilised pH gradients improve membrane proteomics -- Affects of tumor-induced inflammation on membrane proteins abundance in the mouse liver -- Affects of tumor-induced inflammation on biochemical pathways in the mouse liver -- General discussion -- References. / The aim of this thesis was to develop a proteomics methodology that improves the identification of membrane proteomes from mammalian liver. Shotgun proteomics is a method that allows the analysis of proteins from cells, tissues and organs and provides comprehensive characterisation of proteomes of interest. The method developed in this thesis uses separation of peptides from trypsin digested membrane proteins by immobilised pH gradient isoelectric focusing (IPG-IEF) as the first dimension of two dimensional shotgun proteomics. In this thesis, peptide IPG-IEF was shown to be a highly reproducible, high resolution analytical separation that provided the identification of over 4,000 individual protein identifications from rat liver membrane samples. Furthermore, this shotgun proteomics strategy provided the identification of approximately 1,100 integral membrane proteins from the rat liver. The advantages of using peptide IPG-IEF as a shotgun proteomics separation dimension in conjunction with label-free quantification was applied to a biological question: namely, does the presence of a spatially unrelated benign tumor affect the abundance of mouse liver proteins. IPG-IEF shotgun proteomics provided comprehensive coverage of the mouse liver membrane proteome with 1,569 quantified proteins. In addition, the presence of an Englebreth-Holm-Swarm sarcoma induced changes in abundance of proteins in the mouse liver, including many integral membrane proteins. Changes in the abundance of liver proteins was observed in key liver metabolic processes such as fatty acid metabolism, fatty acid transport, xenobiotic metabolism and clearance. These results provide compelling evidence that the developed shotgun proteomics methodology allows for the comprehensive analysis of mammalian liver membrane proteins and detailed some of the underlying changes in liver metabolism induced by the presence of a tumor. This model may reflect changes that could occur in the livers of cancer patients and has implications for drug treatments. / Mode of access: World Wide Web. / 609 p. ill. (some col.)

Es wird allgemein angenommen, dass Proteine exponentiell degradiert werden. Das bedeutet, dass neu synthetisierte als auch alte Proteine mit gleicher Wahrscheinlichkeit degradiert werden. Es tauchen jedoch immer mehr Hinweise dafür auf, dass das nicht immer der Fall sein muss. Um diese Fragestellung systematisch anzugehen, haben wir eine Methode zur metabolischen Pulsmarkierung mit der nichtkanonischen Aminosäure Azidohomoalanine (AHA) entwickelt. AHA ermöglicht die Anreicherung von neu synthetisierten Proteinen direkt nach einem Puls oder nach einer „chase“ (Nachverfolgung) Periode in AHA freiem Medium. Wir kombinierten diese Methode mit SILAC und Shotgun Proteomik um zu quantifizieren wieviel Protein nach verschiedenen chase-Perioden übrig bleibt. Damit konnten wir Degradationsprofile für tausende von Proteinen erstellen. Unsere Daten zeigen, dass mehr als 10 % der Proteine nicht exponentiell degradiert werden (NED). Diese Proteine werden mit fortschreitendem Alter ausschließlich stabiler. Proteasomale Degradation von überschüssigen Proteinkomplexuntereinheiten scheint einen Großteil der NEDs zu erklären. Beim Vergleich zwischen murinen und humanen Zellen stellte sich heraus, dass NED teilweise konserviert ist. Das liegt scheinbar daran, dass diese Zellen trotz unterschiedlichem Ursprungs einheitlich bestimmte Untereinheiten überproduzieren. Da überschüssige NED Proteine bereits unter Standardbedingungen degradiert werden, nahmen wir an, dass die zusätzliche Überproduktion eines NED Proteins seine Level im stationären Zustand nicht verändern sollte. Um dies zu zeigen, quantifizierten wir Degradationskinetiken von Proteinen einer aneuploidenZelllinie. Wir fanden, dass NED Proteine, die auf trisomischen Chromosomen codiert sind, nicht in gleichem Maße ihr stationäres Level steigerten wie exponentiell degradierte Proteine. In Übereinstimmung mit unserer Hypothese verzeichneten wir stattdessen eine Zunahme der anfänglichen Degradationsraten dieser NED Proteine. / Proteins are thought to be degraded exponentially. That means that newly synthesized proteins have the same probability to be degraded as old proteins. However, evidence has accumulated showing that this is not true in all cases. To analyze this more systematically, we developed a method employing metabolic pulse-labeling by the non-canonical amino acid azidohomoalanine (AHA). AHA enables enrichment of newly synthesized proteins directly after pulse or after chase in AHA-free medium. We used SILAC and shotgun proteomics to quantify how much protein remains after different lengths of chase to create degradation profiles for thousands of proteins. Importantly, these degradation profiles allowed us to detect changes in degradation kinetics as the proteins age. We found that more than 10 % of proteins are non-exponentially degraded (NED). These protein are exclusively stabilized by age. Proteasomal degradation of excess protein complex subunits seems to explain a large fraction of NED. Comparing NED in mouse and human cells, we found that NED is at least partially conserved, seemingly due to cells consistently making too much of certain subunits. These overproduced subunits are on average shorter and more structured than the exponentially degraded proteins within the same complex. Finally, since excess NED proteins are degraded during baseline conditions, we hypothesized that making more of a NED protein would not increase its steady state levels. We employed an aneuploidy cell model and found that indeed NED proteins encoded on trisomic chromosomes did not increase in steady state levels to the same extent as exponentially degraded proteins. Instead, we recorded an increase in initial degradation of these proteins. In summary, we present a method for global pule-chase experiments allowing the detection of age-dependent protein degradation with possible implications for the understanding of aneuploidy and cancer.

Das Biopolymer-produzierende Knallgasbakterium Ralstonia eutropha H16 gilt mit seinem außergewöhnlichen Stoffwechsel als vielversprechender Produktionsstamm für die weiße Biotechnologie. Es wächst auf einer Vielzahl organischer Substrate sowie chemolithoautotroph mit H2 und CO2 als einzige Energie- bzw. Kohlenstoffquelle. Unter anaeroben Bedingungen ist es zudem zur Denitrifikation befähigt. In dieser Arbeit wurde das Proteinprofil von R. eutropha unter chemolithoautotrophen sowie anaeroben Bedingungen mittels GeLC-MS/MS untersucht. Beide Proteomstudien offenbarten, dass die Nutzung unterschiedlicher Elektronendonoren bzw. -akzeptoren mit zahlreichen Veränderungen im Proteinbestand der Zellen einherging. Hierbei waren neben Proteinen metabolischer und Transportprozesse auch jene der Zellbewegung betroffen. Die Ergebnisse stellen im Vergleich zu vorangegangenen Studien den bisher umfassendsten Überblick zum Proteinbestand beim H2-basierten sowie anaeroben Wachstum in R. eutropha dar. Von besonderer Bedeutung war dabei das Einbinden der Analyse der Membran als Ort wichtiger Energie- und Transportprozesse. Besonderes Interesse galt einem unter H2/CO2-Bedingungen abundanten Zweikomponentensystem. Sequenzvergleiche zeigten Ähnlichkeit zum Regulationssystem der Katabolitrepression des Biphenylabbaus in Acidovorax sp. KKS102. Die Deletion des Response-Regulator-Gens führte zu vielfältigen Wachstumseffekten auf Substraten wie Fructose, Glycerin sowie auf H2/CO2. Der pleiotrope Phänotyp sowie die Ergebnisse von Genexpressionsstudien und der Suche nach Regulator-Bindestellen lassen eine globale Rolle des Systems im Energie- und/oder Kohlenstoffmetabolismus von R. eutropha H16 annehmen. Histidin-Kinase und Response Regulator wurden in GloS bzw. GloR umbenannt. Die vorliegende Arbeit zeigt eindrucksvoll das Potential der Proteomik als Teil der funktionellen Genomik für den Anstoß neuer Forschungsansätze zur Evaluierung des biotechnologischen Potentials von Mikroorganismen. / Due to its remarkable metabolism the bioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16 is ranked as a promising production strain for white biotechnology. It grows on a wide range of organic substrates as well as lithoautotrophically on H2 and CO2 as sole energy and carbon source, respectively. Under anaerobic conditions it thrives by denitrification. This thesis focused on characterizing the protein profiles of lithoautotrophically and anaerobically grown R. eutropha cells. Proteome analyses revealed an extensive protein repertoire adapting the organism to alternative electron donors and acceptors, respectively. Changes concerned proteins involved in metabolic and transport processes as well as in cell movement. Compared to previous studies the results reported here offer the most comprehensive proteomic survey regarding the H2-based as well as anaerobic lifestyle of R. eutropha so far. In this context analyzing the cell membrane as a place for a number of energy, transport and signal transduction processes was of particular importance. Special interest aroused the identification of a two-component system upregulated on H2/CO2. Sequence analysis offered high similarity to the regulatory system for catabolite control of biphenyl degradation in Acidovorax sp. KKS102. Deletion of the response regulator gene led to versatile growth effects on substrates such as fructose and glycerol as well as H2/CO2. This pleiotrophic phenotype as well as the results of gene expression studies and the search for regulator binding sites suggests that the two-component system is a global player in energy and/or carbon metabolism in R. eutropha and possibly other bacteria. Thus, histidine kinase and response regulator have been renamed GloS/R. Since their characterization was initiated by proteomic data this study impressively elucidates the power of functional genomics in terms of revealing new research approaches to evaluate the biotechnological use of microbes.

Proteins and their intricate network of interactions are fundamental to many molecular processes that govern life. Mass spectrometry-based structural proteomics represents a powerful set of techniques for characterizing protein structures and interactions. The last decade has witnessed a large-scale adoption in the application of these techniques toward solving a variety of biological questions. Addressing these questions has often been coincident with the further development of these techniques. Insight into the structures of individual proteins and their interactions with other proteins in a proteome-wide context has been made possible by recent developments in the relatively new field of chemical crosslinking combined with mass spectrometry. In these experiments crosslinking reagents are used to capture protein-protein interactions by forming covalent linkages between proximal amino acid residues. The crosslinked proteins are then enzymatically digested into peptides, and the covalently-coupled crosslinked peptides are identified by mass spectrometry. These identified crosslinked peptides thus provide evidence of interacting regions within or between proteins. In this dissertation the development of tools and methods that facilitate this powerful technique are described. The primary arc of this work follows the development and application of mass spectrometry-based approaches for the identification of protein crosslinks ranging from those which exist endogenously to those which are introduced synthetically. Firstly, the development of a novel strategy for comprehensive determination of naturally occurring protein crosslinks in the form of disulfide bonds is described. Secondly, the application of crosslinking reagents to create synthetic crosslinks in proteins coupled with molecular dynamics simulations is explored in order to structurally characterize the intrinsically disordered tau protein. Thirdly, improvements to a crosslinking-mass spectrometry method for defining a protein-protein interactome in a complex sample is developed. Altogether, these described approaches represent a toolset to allow researchers to access information about protein structure and interactions. / Graduate