The role of histone modifications in epigenetic regulation
Post-translational modifications of histone proteins play key roles in modulating gene activation and silencing and are involved in mediating epigenetic inheritance of those effects. In recent years it has become evident that these processes are controlled by combinations of coexisting covalent modifications that are primarily located on histone tails.
Our goal is to develop a mass spectrometry based strategy that will allow us to identify, locate in protein sequence and quantify the abundance of various covalent modifications, namely: acetylation, methylation, ubiquitination and phosphorylation.
Our approach is based on middle-down proteomics strategy. It utilizes the optimized performance of hybrid resins based liquid chromatography separation of large peptides generated from histone partial digestion, and ETD based mass spectrometric peptide sequencing, that is specifically suited for fragmentation of large peptides containing combinations of covalent modifications. This strategy combined with bioinformatics tools tailor-made for analysis of data generated from middle down type proteomics experiments allows us currently to characterize in a high-throughput manner multiply modified histone tails of 40-70 amino acids.
The method is applied to different biological models relevant for studying epigenetic regulation of gene activation and silencing.
Simone Sidoli, Veit Schwämmle, Simon Haldbo, Adelina Rogowska-Wrzesinska, Ole N. Jensen
Psoriasis: Proteomic Characterisation and Biomarker Discovery
Psoriasis affects 2-3% of the world’s population and can have a severe impact on quality of life. The aim of this project is to gain a better understanding of the disease and also to uncover biomarkers that can be used to predict therapeutic responses to the wide variety of pharmaceuticals available for treatment. This project primarily involves academic research at SDU, but also includes industrial and clinical collaboration.
James Williamson, Adelina Rogowska-Wrzesinska, Ole Nørregaard Jensen
Characterising the ‘Modificome’ of Developing Brains
We aim to characterise the post-translational modification (PTM) status of the proteome of the mouse brain over a developmental time-course. Many PTMs (e.g. glycosylation, phosphorylation, acetylation…) are known to influence protein structure and function in profound ways, and it is our hypothesis that describing PTM patterns and changes in these patterns will reveal new insights into how brain development, an enormously complex process, is controlled on a molecular level. We subsequently aim to apply molecular biology techniques to specific instances of PTM in development to further describe the functional effects of particular types of PTM.
We are also looking at the phenomenon of large-scale PTM coding, whereby complex patterns of PTMs may be used by cells to carry information. This would allow an enormous increase in the complexity of cellular systems, and is an appealing mechanism to explain at least some proportion of the finely controlled regulation seen in living organisms. This will be described using tools developed in-house, as well as tested using targeted protein chemistry and mass spectrometric approaches.
Participants: Alistair V. G. Edwards, Martin R. Larsen.
Development of proteomics methods for investigation of oxidised and membrane bound proteins
Protein oxidation occurs naturally in aerobic organisms. Reactive oxygen and nitrogen species (ROS and RNS) modify proteins altering their structure and function thus affecting molecular pathways which they are involved in. Under homeostatic conditions protein oxidation is believed to play a crucial role in cell signaling (i.e. thiol oxidation). Under severe oxidative stress (disease, aging) cellular level of ROS and RNS rise rapidly leading to protein carbonylation - an irreversible modification.
Investigating mechanisms of action and characterisation of oxidised proteins is crucial for a better understanding of molecular mechanisms of oxidative stress-related diseases (i.e. diabetes mellitus, Alzheimer’s disease).
The overall goal of this project is to develop and optimise new strategies for identification and characterisation of protein oxidation, thiol oxidation and carbonylation in particular. We combine chemical derivatization, affinity enrichment and state-of-the art mass spectrometry to achieve set goals.
This project is a joined collaboration with Proteomics Group at Beijing Institute of Genomics, Chinese Academy of Sciences established within Sino-Danish Center for Education and Research.
Participants: Katarzyna Wojdyla, (James Williamson), Adelina Rogowska-Wrzesinska, Peter Ropstorff (PR Group) and Siqi Liu (Proteomics Group, Beijing Institute of Genomics, Chinese Academy of Sciences)
Influence of Sialic Acid Modifcation of Surface Proteins on Intracellular Signalling
Post-translational modifications (PTMs) of proteins refer to the addition of chemical modifying groups to proteins after their synthesis. PTMomics is a relatively new research area aiming at understanding the function and consequences of PTMs of proteins in a cell. Recently, it has been discovered that changes to a particular PTM on a protein can have a significant influence on other PTMs which in turn can alter the function or activity of proteins. Previously, phosphorylation, O-GlcNAcylation and acetylation have been proposed to interplay with each other in regulation of signalling pathways initiated by external stimuli; however the exact mechanisms and consequences are poorly studied. Recently, we have discovered that sialylation of cell-surface glycoproteins has the potential to influence intracellular phosphorylation–dependent signalling. The aim of this project is to develop an analytical strategy for identification and quantification of intact sialylated glycopeptides from cell-surface glycoproteins and apply this strategy to characterize the influence of sialylation of these proteins on intracellular phosphorylation-dependent signalling pathways. Especially, the effect of modulation of sialylation of the pathways involved in epidermal growth factor signalling and calcium mediated release of neurotransmitters from nerve endings will be studied. A thorough understanding of the complex cross-talk between sialylation and phosphorylation is needed, in order to be able to understand the true complexity and diversity of cellular signalling initiated by external stimuli in health and disease samples.
Maria Ibañez-Vea
An Interregional and Interdisciplinary Joint Venture for Comprehensive Characterization of Nanoparticles and their Cellular Effect on Human Cell Lines
The use of nanoparticles (NP) in the food industry has evolved dramatically during the last few years. This can be explained by the wide range of applications of nanoparticles facilitating enhanced flavour, texture, solubility and preservation features in food. Because of this development and the increasing exposure of humans to nanoparticles, which will probably increase in the future, there is an urgent need to investigate the health risks arising from the use of NP in food. However, this task in itself is very complex as its study is highly interdisciplinary. The aim of this collaboration is to achieve a comprehensive assessment of the health risk involved in the exposure of human cells to NP. Three different research groups (Protein Research Group, Odense, SDU; Biotechnology Research Facility, Flensburg, University of Applied Sciences Flensburg; and Nano-Technology, Sonderborg, SDU) and an industrial partner (KIN Food Institute, Neumünster) are involved in this project.
Our group is performing a mass spectrometry-based quantitative proteome study of the human gastrointestinal LOVO cells treated with NP in order to detect proteins differentially expressed during the exposure.
Frank Kjeldsen, Karin Hjernø, Thiago Verano Braga, Veit Schwämmle
CrossWork: A Bioinformatic Approach to Identifying Peptides with Complex Modifications from MS Datasets
Most common search engines for analysing mass-spectrometric datasets keep a strict focus on identifying the peptides/proteins in the sample, while downplaying the importance of identifying peptide modification(s).
We work with samples where the protein composition is largely known, but which may contain several types of modifications (natural or chemically induced), including cross-linked peptides, glycosylations, multiple variable modifications, hydrogen-deuterium exchange labeling, non-specific (bacterial) digests, etc.: features that are poorly catered for - if at all - by most MS analytical software.
We have therefore developed a series of software tools in order to facilitate analysis of these and more. Through developing our own analytical software, we can continuously meet new challenges to MS analysis as they arise, while reducing the reliance on third party tools over which we have little influence.
The CrossWork suite comprises the programs: CrossWork, Glycanthrope and MassAI, all of which can be downloaded from www.proteomics.dk
Morten I Rasmussen, Thomas JD Jørgensen, Peter Højrup.
Retrograde signalling from mitochondria by oxidized peptides
Reactive oxygen species (ROS) can react with and irreversibly damage cellular components. Each cell compartment contains enzymes designed to limit ROS accumulation, but under stress these enzymes might be overwhelmed and ROS accumulates. To prevent this, as yet unknown, signal transduction mechanisms trigger a programme of gene expression to maintain ROS homeostasis.
We have proposed that there is a special signalling system in eukaryotic cells in which oxidized peptides deriving from proteolytic breakdown of oxidatively damaged proteins, e.g., in mitochondria, act as specific messengers to regulate source-specific genes. Our aim is to test this hypothesis.
Jesper F. Havelund, Adelina Rogowska-Wrzesinska, Ole N. Jensen together with Jay J. Thelen from University of Missouri, Columbia, Lee J. Sweetlove University of Oxford, UK and Ian Max Møller from Aarhus University, Denmark
Nutriefficient
Sustainable improvements in the future productivity of cropping systems depend on novel resource-efficient plant genotypes being designed to match site-specific soil and climatic conditions. NUTRIEFFICIENT aims to deliver new technologies for the efficient selection and breeding of plant genotypes with improved nutrient-use efficiency.
Jørgen Petersen, Adelina Rogowska-Wrzesinska, Ole N. Jensen together with Jan Kofod Scjhørring from University of Copenhagen, Denmark and other project partners
Proteomic analysis of stimulated neutrophils
Neutrophils are the most abundant white blood cells: they play a major role in inflammatory and immunological responses. They are the first cells to arrive at the tissue in response to alesionor invasion of microorganisms . Neutrophils are responsible for the phagocytosis of microorganisms and tissue debris and also for releasing molecules that will stimulate other cells, thus promoting the organism defense system. Although neutrophils are important as a defense, their actions can also cause damage. In a severe trauma, the inflammatory response escapes its normal regulation, and activated neutrophils release toxic molecules outside the affected tissue, causing clinical conditions with high mortality such as the Acute Respiratory Distress Syndrome or the Multiple Organ Disfunction Syndrome.
The aim of this project is to understand how the neutrophils escape the normal regulation and to try to prevent or reverse this response by analyzing the proteomic profile of this cell type under different stimulation conditions, both in vivo and in vitro. With such data we hope to fill in the blanks in the understanding of the activation pathways and possibly propose targets for a better control of the process.
From Brazil: Wagner Fontes, Mariana S. Castro, Elaine N. Aquino, Anne C.D. Neves, Micaella P. Fonseca, Belchor Fontes, Muhammad Tahir, Samina Arshid, Pollyanna B. F. Correa.
From SDU: Peter Roepstorff, Richard Sprenger
Biomarker LC project
The focus of this project is on the development of new short / fast LC-SRM methods to quantify known biomarkers in biological fluids (blood, urine, ...) to be implemented in a new LC prototype (SPELC) from Thermo Scientific: a standard easy-LC instrument equipped with a new autosampler that connects a stageTip (typically with a C18 disk) to the column (i.e. using the tip as a precolumn).
Clear advantages of this approach are: 1) one-time use of the precolumn (no carryover effects, longer life-time of the column) and 2) off-line equilibration, loading and washing of the precolumn (leading to reduced analysis time, a greater number of samples can be analysed per day).
From SDU: Gerard Such, Adelina Rogowska-Wrzesinska, Ole N. Jensen
From Thermo Scientific: Nicolai Bache
Investigation of transcription factors and other proteins involved in early adipogenesis by using genomic and proteomic approaches
Adipocytes play a key role in the development of obesity and diabetes. The transcription factor C/EBPβ is known to be required for adipocyte differentiation both in vitro and in vivo. It facilitates the binding of early adipogenic transcription factors to chromatin. The main focus of the project is to investigate the role of C/EBPβ and associated transcription factors and co-factors in chromatin remodeling in early adipocyte differentiation.
- We combine immunoprecipitation (IP) with quantitative proteomics (LC-ESI-MS/MS) to identify protein complexes.
- We combine chromatin immunoprecipitation with deep sequencing (ChIP-seq) to investigate the chromatin binding sites of transcription factors.
- We study the functional role of selected proteins using knockdown/overexpression experiments.
Atefeh Rabiee, Simone Sidoli, Rasmus Siersbæk, Adelina Rogowska- Wrzesinska, Susanne Mandrup, Ole Nørregaard Jensen.
Urinary exosomic proteins as biomarkers in syndromes involving kidney function
The aim of this project is to use urinary exosomic proteins to obtain biomarker signatures for diseases with kidney involvement (hypertension, graft rejection) and address hypotheses relating to the epithelial sodium channel, ENaC, in conditions with proteinuria and hypertension. The perspectives are to develop - ultimately to the dipstick level - more sensitive, non-invasive biomarker methods with diagnostic and prognostic potentials, in kidney-related diseases; and secondly, to gain mechanistic information on the pathophysiology behind hypertension in proteinuric diseases.
Pia Hønnerup Jensen, Peter Bie (Institute of Molecular Medicine) and Ole Nørregaard Jensen, in collaboration with several departments at Odense University Hospital.
Metal Tags for Proteomics
Mass spectrometry is a powerful technique to study changes in the protein expression of cells in response to stimuli. Especially the investigation of post-translational modifications (PTMs) is highly interesting. Some PTMs are, however, inherently difficult to study with mass spectrometry. The most common PTM, phosphorylation, is for example often lost during MS/MS when subjected to vibrational excitation. This interdisciplinary project aims at developing bioinorganic metal complexes or “tags” for different functional groups found in biochemically interesting molecules, such as proteins, DNA and lipids. The tags must be highly specific towards their targets and they must help improve the information gained from MS and MS/MS investigations. In the case of protein phosphorylation we have recently developed a tag which stabilizes the phosphate ester bond and thereby inhibiting the loss of this modification.
Simon Svane, Fedor Kryuchkov, Thiago Verano Braga, Christine J. McKenzie (FKF) and Frank Kjeldsen
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