Background: The unicellular green alga Chlamydomonas reinhardtii is an important eukaryotic model organism for the study of photosynthesis and plant growth. In the era of modern highthroughput technologies there is an imperative need to integrate large-scale data sets from highthroughput experimental techniques using computational methods and database resources to provide comprehensive information about the molecular and cellular organization of a single organism. Results: In the framework of the German Systems Biology initiative GoFORSYS, a pathway database and web-portal for Chlamydomonas (ChlamyCyc) was established, which currently features about 250 metabolic pathways with associated genes, enzymes, and compound information. ChlamyCyc was assembled using an integrative approach combining the recently published genome sequence, bioinformatics methods, and experimental data from metabolomics and proteomics experiments. We analyzed and integrated a combination of primary and secondary database resources, such as existing genome annotations from JGI, EST collections, orthology information, and MapMan classification. Conclusion: ChlamyCyc provides a curated and integrated systems biology repository that will enable and assist in systematic studies of fundamental cellular processes in Chlamydomonas. The ChlamyCyc database and web-portal is freely available under http://chlamycyc.mpimp-golm.mpg.de.

Malignant glioma consists of a group of diseases where the localisation and the nature of the disease makes treatment an extreme challenge. Two important biological features of malignant glioma cells are their infiltrative growth and their ability to induce angiogenesis. Glioma cells migrate extensively behind the blood-brain barrier and infiltrate the surrounding brain making radical treatment with surgery and radiotherapy almost impossible. The aims of this thesis were to investigate factors of importance for glioma cell migration and angiogenesis and to evaluate if an anti-angiogenesis approach alone or in combination with current treatment modalities could inhibit glioma growth. For this purpose we used the BT4C orthotopic rat glioma model and investigated treatment effects of the vascular endothelial growth factor (VEGF) receptor-2 and epidermal growth factor (EGF) receptor tyrosine kinase inhibitor ZD6474 alone or in combination with temozolomide or radiotherapy. Altered protein expression pattern after anti-angiogenesis treatment was measured using a mass-spectrometric proteomic method, followed by multivariate data-analysis. The tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and VEGF showed altered temporal and spatial mRNA expression during glioma progression. In early stages of tumour progression the expression was found throughout the tumour while in later stages, the expression was more predominant in the invasive tumour border. ZD6474 in monotherapy significantly inhibited tumour growth in the BT4C glioma model. The effect was further enhanced when combined with radiotherapy or temozolomide. Using mass-spectrometric methods an altered protein expression pattern after ZD6474 treatment was observed implicating the possibility to use proteomic methods for finding predictive biomarkers for anti-angiogenesis treatment. In conclusion, this thesis demonstrates a co-expression of factors important for glioma growth and angiogenesis and that treatment with an angiogenesis inhibitor has additive effects on glioma growth when combined with radiotherapy and chemotherapy. Finally, an altered protein expression pattern after anti-angiogenesis treatment is evident and detectable. Hopefully this work will contribute to and encourage further research to reach a better understanding of how to combine and evaluate different treatment approaches in malignant glioma.

Peroxisome is a ubiquitous eukaryotic organelle with a single-layer membrane. It maintains various functions that differ depending on the species and cell types, as well as the environmental or developmental conditions. In the first part of this thesis, the peroxisomal protein content was systematically analyzed in different organs in mouse from different ages using proteomic approaches. Thirty-one peroxisomal proteins were identified and ten putative peroxisomal proteins were suggested. The results indicate that peroxisomal proteins show a tissue-specific functional response to the aging process that is probably dependent on their differential regeneration capacity. Besides, alteration in the fatty acid metabolism could alter membrane protein functions; decrease in catalase expression in kidney may contribute to oxidative stress and isoprenoid biosynthesis could contribute to decline in bile salt synthesis. The ability to detect changes in the peroxisomal proteome associated with organ impairment during the course of aging would provide a conceptual framework to understand the role of peroxisome in aging. In the second part, peroxisome proteomics was used as a novel approach in marine pollution assessment. The peroxisomal protein expression profiles were obtained and identified from mussel Mytilus sp. exposed to different pollutants, in both laboratory and field experiments. The identified proteins were involved in α- and β–oxidation pathways, xenobiotics and amino acid metabolism, cell signalling, oxyradical metabolism, peroxisomal assembly, respiration and cytoskeleton pathway, etc. Generally, these findings suggest that protein expression signatures could become a valuable tool to monitor the presence of pollutants in marine environment.

Type 2 diabetes mellitus (T2DM) is a heterogeneous disease affecting millions of people worldwide. Both genetic and environmental factors contribute to the pathogenesis. The disease is characterized by alterations in many genes and their products. Historically, genomic alterations have mainly been studied at the transcriptional level in diabetes research. However, transcriptional changes do not always lead to altered translation, which makes it important to measure changes at the protein level. Proteomic techniques offer the possibility of measuring multiple protein alterations simultaneously. In this thesis, the proteomic technique surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS) has been applied and evaluated in the context of T2DM research. Protocols for pancreatic islet and serum/plasma protein profiling and identification have been developed. In addition, the technique was used to analyze the influence of genetic background versus diabetic environment by determining serum protein profiles of individuals with normal glucose tolerance (NGT) and T2DM with or without family history of diabetes. In total thirteen serum proteins displayed different levels in serum from persons with NGT versus patients with T2DM. Among these proteins, apolipoprotein CIII, albumin and one yet unidentified protein could be classified as being changed because of different genetic backgrounds. On the other hand, ten proteins for instance transthyretin, differed as a result of the diabetic environment. When plasma protein patterns of NGT and T2DM individuals characterized by differences in early insulin responses (EIR) were compared, nine proteins were found to be varying between the two groups. Of these proteins five were identified, namely two forms of transthyretin, hemoglobin α-chain, hemoglobin β-chain and apolipoprotein H. However no individual protein alone could explain the differences in EIR. In conclusion, SELDI-TOF MS has been successfully used in the context of T2DM research to identify proteins associated with family history of diabetes and β-bell function.

In this thesis, methods to study gene and protein expression in cells and tissues were developed and utilized in combination with protein-specific antibodies, with the overall objective to attain greater understanding of protein function. To analyze protein expression in in vitro cultured cell lines, a cell microarray (CMA) was developed, facilitating antibody-based protein profiling of cell lines using immunohistochemistry (IHC). Staining patterns in cell lines were analyzed using image analysis, developed to automatically identify cells and immunohistochemical staining, providing qualitative and quantitative measurements of protein expression. Quantitative IHC data from CMAs stained with nearly 3000 antibodies was used to evaluate the adequacy of using cell lines as models for cancer tissue. We found that cell lines are homogenous with respect to protein expression profiles, and generally more alike each other, than corresponding cancer cells in vivo. However, we found variability between cell lines in regards to the level of retained tumor phenotypic traits, and identified cell lines with a preserved link to corresponding cancer, suggesting that some cell lines are appropriate model systems for specific tumor types. Specific gene expression patterns were analyzed in vitiligo vulgaris and malignant melanoma. Transcriptional profiling of vitiligo melanocytes revealed dysregulation of genes involved in melanin biosynthesis and melanosome function, thus highlighting some mechanisms possibly involved in the pathogenesis of vitiligo. Two new potential markers for infiltrating malignant melanoma, Syntaxin-7 and Discs large homolog 5, were identified using antibody-based protein profiling of melanoma in a tissue microarray format. Both proteins were expressed with high specificity in melanocytic lesions, and loss of Syntaxin-7 expression was associated with more high-grade malignant melanomas. In conclusion, the combination of antibody-based proteomics and microarray technology provided valuable information of expression patterns in cells and tissues, which can be used to better understand associations between protein signatures and disease.

The primary function of adipose tissue is to store energy in the form of triacylglycerol, which is hydrolyzed to fatty acids to supply other tissues with energy. While insulin promotes the storage of triacylglycerol, catecholamines stimulate its hydrolysis. The development of type II diabetes is strongly associated with obesity, indicating a role of triacylglycerol metabolism in the pathogenesis of diabetes. Caveolae are plasma membrane invaginations found in most cells but are highly abundant in adipocytes. Insulin receptors are localized in caveolae and their function depends on intact caveolae structures. In the present thesis work, mass spectrometry-based methodology allowed identification of a number of new proteins and their posttranslational modifications in caveolae of human adipocytes. Variable N-terminal acetylation and phosphorylation of caveolin-1α and caveolin-1β were identified, which might regulate the function of caveolae. The transcription regulator protein PTRF was identified as the major caveolae associated protein. Specific proteolytic modifications of PTRF at the cytosolic surface of caveolae and phosphorylation on nine serine and one threonine residues were identified. Moreover, insulin induced translocation of PTRF from the plasma membrane to the nucleus. PTRF was previously shown to regulate the activity of both RNA polymerase I and polymerase II, thus a role of PTRF in mediating the anabolic action of insulin on protein synthesis and gene transcription is proposed. PTRF was also involved in an extranuclear function in the hormonal regulation of triacylglycerol metabolism in caveolae. PTRF was colocalized with the triacylglycerol regulator proteins perilipin and hormone-sensitive lipase (HSL) in the triacylglycerol-synthesizing caveolae subclass. We showed that, while perilipin was translocated to the plasma membrane, both PTRF and HSL were translocated from the plasma membrane to the cytosol as a complex in response to insulin. The perilipin recruited to the plasma membrane was highly threonine phosphorylated. By mass spectrometry, three phosphorylated threonine residues were identified and were located in an acidic domain in the lipid droplet targeting domain of perilipin. The insulin-induced recruitment of perilipin to the plasma membrane might, therefore be phosphorylation-dependent. Isoproterenol, which stimulates hydrolysis of triacylglycerol, induced a complete depletion of perilipin B from the plasma membrane, suggesting a function of perilipin B to protect newly synthesized triacylglycerol in caveolae from being hydrolyzed by HSL. The location of PTRF and HSL was not affected by isoproterenol, indicating that insulin is acting against a default presence of PTRF and HSL in caveolae. Taken together, this thesis expands our knowledge about caveolae and provided valuable information about their involvement in novel roles, particularly in the hormonal regulation of triacylglycerol metabolism.

The aim of this thesis is to provide a thorough characterization of the photosynthetic machinery from the heterocysts of Nostoc punctiforme strain ATCC 29133. In this thesis I describe the protocols I have optimized for the isolation of thylakoids from vegetative cells, the purification of heterocysts and the isolation of thylakoids from the purified heterocysts. The composition of the thylakoid membranes was studied by two dimensional electrophoresis and mass-spectrometry. Further insight into the functionality of the photosynthetic complexes was obtained by EPR, electron transport measurements through Photosystem II (PSII), and fluorescence spectroscopy. The proteome of the heterocysts thylakoids compared to that of the vegetative cell was found to be dominated by Photosystem I (PSI) and ATP-synthase complexes, both essential for keeping high nitrogenase activities. Surprisingly, we found a significant amount of assembled monomeric PSII complexes in the heterocysts thylakoid membranes. We measured in vitro light-driven electron transfer from PSII in heterocysts using an artificial electron donor, suggesting that under certain circumstances heterocysts might activate PSII. Parallel to my main research I also worked in a collaboration to elucidate the total proteome of Nostoc sp. strain 7120 and Nostoc punctiforme using quantitative shotgun proteomics. Several hundred proteins were quantified for both species. It was possible to trace the detailed changes that occurred in the energy and nitrogen metabolism of a heterocyst after differentiation. Moreover, the presence of PSII proteins identified in our membrane proteome was also confirmed and extended. Lastly, I studied how the heterocysts are capable of responding to variations in light quality as compared to vegetative cells. Using 77 K fluorescence spectroscopy on heterocysts and vegetative cells previously illuminated with light at specific wavelengths, I was able to demonstrate that heterocysts still possess a possibly modified but functional antenna system, capable of harvesting light and transferring energy preferentially to PSI. The characterization of the membrane and total proteome permitted to draw a more comprehensive and integrated picture of the interplay between the distinct metabolic processes that are carried out in each cell type at the same time; from oxygenic photosynthesis and carbon fixation in the vegetative cells to the anoxygenic cyclic photophosphorylation essential to power nitrogen assimilation in the heterocysts.

Biological function is mainly carried out by a dynamic population of proteins which may be used as markers for disease diagnosis, prognosis, and as a guide for effective treatment. In analogy to genomics, the study of proteins is called proteomics and it is generally performed by two-dimensional gel electrophoresis and mass spectrometric methods. However, gel based proteomics is methodologically restricted from the low mass region which includes important endogenous peptides. Furthermore, the study of endogenous peptides, peptidomics, is compromised by protein fragments produced post mortem during conventional sample handling. In this thesis nanoflow liquid chromatography and mass spectrometry have been used together with improved methods for sample preparation to semi-quantitatively monitor peptides in brain tissue. The proteolysis of proteins and rise of fragments in the low mass region was studied in a time-course study up to ten minutes, where a potential marker for sample quality was found. When rapidly denatured brain tissue was analyzed, the methods enabled detection of hundreds of peptides and identifications of several endogenous peptides not previously described in the literature. The identification process of endogenous peptides has been improved by creating small targeted sequence collections from existing databases. In applications of the MPTP model for Parkinson’s disease the protein and peptide expressions were compared to controls. Several proteins were significantly changed belonging to groups of mitochondrial, cytoskeletal, and vesicle associated proteins. In the peptidomic study, the levels of the small protein PEP-19 was found to be significantly decreased in the striatum of MPTP administered animals. Using imaging mass spectrometry the spatial distribution of PEP-19 was found to be predominant in the striatum and the levels were concordantly decreased in the parkinsonian tissue as verified by immunoblotting.

This thesis describes generation and characterization of Affibody molecules with future applications in proteomics research, protein structure determinations, therapeutic treatment of disease and medical imaging for in vivo diagnostics. Affibody molecules are engineered affinity proteins developed by combinatorial protein engineering from the 58-residue protein A-derived Z domain scaffold. Novel Affibody molecules targeting human proteins were selected from a combinatorial library using phage display technology. In the first two investigations, an Affibody molecule specifically targeting the high abundant human serum protein transferrin was generated. The intended future use of this Affibody ligand would be as capture ligand for depletion of transferrin from human samples in proteomics analysis. Strong and highly specific transferrin binding of the selected Affibody molecule was demonstrated by biosensor technology, dot blot analysis and affinity chromatography. Efficient Affibody-mediated depletion of transferrin in human plasma and cerebrospinal fluid (CSF) was demonstrated in combination with IgG and HSA removal. Furthermore, depletion of five high abundant proteins including transferrin from human CSF gave enhanced identification of proteins in a shotgun proteomics analysis. Two studies involved the selection and characterization of Affibody molecules recognizing Alzheimer’s amyloid beta (Abeta) peptides. Future prospect for the affinity ligands would primarily be for therapeutic applications in treatment of Alzheimer’s disease. The developed A-binding Affibody molecules were found to specifically bind to non-aggregated forms of Abeta and to be capable of efficiently and selectively capture Abeta peptides from spiked human serum. Interestingly, the Abeta-binding Affibody ligands were found to bind much better to Abeta as dimeric constructs, and with impressive affinity as cysteine-bridged dimers (KD~17 nM). NMR spectroscopy studies revealed that the original helix one, of the two Affibody molecules moieties of the cysteine-bridged dimers, was unfolded upon binding, forming intermolecular β-sheets that stabilized the Abeta peptide, enabling a high resolution structure of the peptide. Furthermore, the Abeta-binding Affibody molecules were found to inhibit Abeta fibrillation in vitro. In the last study, Affibody molecules directed to the interleukin 2 (IL-2) receptor alpha (CD25) were generated. CD25-binding Affibody molecules could potentially have a future use in medical imaging of inflammation, and possibly in therapeutic treatment of disease conditions with CD25 overexpression. The selected Affibody molecules were demonstrated to bind specifically to human CD25 with an apparent affinity of 130-240 nM. Moreover, the CD25-targeting Affibody molecules were found to have overlapping binding sites with the natural ligand IL-2 and an IL-2 blocking monoclonal antibody. Furthermore, the Affibody molecules demonstrated selective binding to CD25 expressing cells. / QC 20100729

Recent advances in biotechnology have generated possibilities to investigate and measure parts of life previously left for believers to explain. Utilizing the same book of recipes, the genome, our cells produce selections of proteins at a time and thereby niche into a multitude of specialized cell types, tissues and organs comprising our body. Knowledge of the precise protein composition in a given organ at normal and disease condition would be of invaluable importance, both for identification of disease causes and the design of new pharmaceuticals, as well as for a deeper understanding of the processes of life. This doctoral thesis describes the start and progress of a visionary project (HPR) to localize all human proteins in our body, with emphasis on the generation and characterization of antibodies used as protein targeting missiles. To facilitate the identification of one human protein in a complex environment like our body, it is of significant importance to have precise and specific means of detection. The first two papers (I-II), describe software developed for generation of monospecific antibodies satisfying such needs, using a set of rules for antigen optimization. Five years after project start a large amount of antibodies with documented characteristics have been generated. The third paper (III), illustrates an attempt to sieve these antibody characteristics to develop a tool, for further improvement of antigen selection, based on the correlation between antigen sequence and amount of specific antibody generated.Having a panel of protein-specific antibodies is a possession of a great value, not only for localization studies, but also as possible target-directed pharmaceuticals. In such cases, knowledge of the precise epitope recognized by the antibody on its target protein, is an important aid, both for understanding its effect as well as unwanted cross-reactivity. Paper (IV) describes the development of a high-resolution method for epitope mapping of antibodies using staphylococcal display. An application of the method is described in the last paper (V) where it is used to map an anti-HER2 monospecific antibody with growth-inhibiting effects on breast cancer cells. The monospecific antibody was fractionated into separate populations and five novel epitopes related to cancer cell growth-inhibition was determined.Altogether these methods are valuable tools for generation and characterization of monospecific antibodies. / QC 20100907