CPR > Research Depts. > Proteomics
Department of Proteomics
This department, headed by Prof. Matthias Mann, consists of three independent research groups in addition to the director's group. Mass Spectrometry for Quantitative Proteomics is headed by Prof. Jesper V. Olsen, Proteomics Technology Development and Application is lead by Assoc. Prof. Michael L. Nielsen and Proteomics and Cell Signaling is headed by Assoc. Prof. Chunaram Choudhary.
The department will make use of recent revolutionary breakthroughs in the technology of mass spectrometry based proteomics. These technologies now make it possible to identify thousands of proteins in a wide variety of proteomes, spanning from prokaryotes to cancer tissues. Recent developments in quantitative proteomics also allow comparison to proteomes after stimulation or drug treatment. Other biomedically important capabilities of proteomics include the quantitative determination of post-translational modifications such as phosphorylation, ubiquitination, acetylation, methylation and many others. Not least, proteomics is also able to determine protein - protein, protein - DNA or protein - drug interactions.
Prof. Jesper Velgaard Olsen: Mass spectrometry for quantitative proteomics
This group, headed by Jesper V. Olsen, will focus on establishing and developing methods for quantitative proteomics. The core technology will be high-resolution mass spectrometry (MS) based on state-of-the-art in high-performance hybrid MS instruments. We plan to use quantitative mass spectrometry in combination with bioinformatics in an integrative Systems Biology approach.
Mass spectrometry is the cornerstone of proteomics and we plan to use the latest generation high-accuracy LTQ Orbitrap mass spectrometer, which is the most versatile MS instrument on the market. The orbitrap analyzer provides high-resolution (R=100,000), high dynamic range (>5000) and mass accuracy (sub ppm) in combination with the fastest scanning ion trap that allow generation of multiple tandem mass spectra (> 5x MS/MS per second) in parallel with orbitrap detection. In the near future the LTQ Orbitrap instrument will also be equipped with the novel peptide fragmentation technique termed electron transfer dissociation (ETD), which allow for pinpointing the position of very labile PTMs (e.g. glycosylations) in modified peptides.
The main focus of the group will be on post-translational modification (PTM) such as protein phosphorylation analyses using high-performance electrospray mass spectrometry in-line with nanoflow HPLC separation (LC-MS/MS). To study phosphorylation-dependent signaling pathways we have previously developed an LC-MS based quantitative phosphoproteomics technology that combines stable isotope labeling by amino acids in cell culture (SILAC) for quantitation with phosphopeptide enrichment and high-performance mass spectrometry. We have recently reported the time-resolved analysis of changes in the phosphoproteome of HeLa cells stimulated with EGF and quantified more than 6000 phosphorylation sites (Olsen et al. Cell 2006).
We will establish our phosphoproteomics platform that will allow us to analyze in vivo protein phosphorylation on a global scale in any cell culture system, and quantify the phosphorylation site dynamics in response to drugs or cytokines/growth factors. We want to further develop this technology and we plan to extend it to study both phosphorylation as well as other PTMs in a way that captures their temporal and spatial distribution in cell signal transduction networks.
Projects planned include studies on the cell division cycle by analyzing cells arrested in different cell cycle stages, DNA-damage repair in response to DNA damaging agents, as well as global analysis of cytokine and growth factor signaling networks implicated in cancer and other diseases.
Prof. Matthias Mann: Proteomics and the Metabolic Syndrome
The Metabolic Syndrome consists of a pattern of morbidities connected with obesity, insulin resistance and cardiovascular abnormalities. It overlaps, but is not exclusive to diabetes. The metabolic syndrome is thought to arise due to lifestyle changes leading to sedentary behavior and obesity. It is one of the greatest emerging public health issues and is thought to overtake any other disease category in associated health cost.
We will apply the power of modern proteomics methods to all aspects of the metabolic syndrome. We have already performed in depth proteomic studies of mitochondria, key players in metabolic diseases. Furthermore, we have studied key peripheral tissues involved in insulin signaling and the metabolic syndrome, such as adipose tissues, muscle tissue and liver.
This work will be pursued in conjunction with other groups in the Copenhagen and Scandinavian area and elsewhere. For example, we already have a collaboration with the Center of Inflammation and Metabolism (CIM) headed by Prof. Bente Petersen. Likewise we are part of the Diabetes Anatomy Project (DGAP), headed by Prof. Ron C. Kahn of the Joslin Diabetes Center at Harvard Medical School.
Michael Lund Nielsen, PhD: Proteomics Technology Development & Application
The focus of the group will be on developing new proteomic technologies and implementing these in relevant biological research areas. While the goals of proteomics are long-standing, the technology to address them is very challenging and is still in development. The group will be heavily involved in this technology development using state-of-the-art, high resolution mass spectrometry. We will aim at continuously improving our proteomic platform, hereby allowing for rapid and comprehensive analysis of mammalian proteomes within a single day. In addition, we will establish a quantitative proteomics platform for unbiased analysis of novel and unexpected PTMs and mutations implicated in the signaling networks of cancer, diabetes and other diseases. Since PTMs predominantly are present in substoichiometric amounts within the cell compared to their non‐modified counterparts, it is advantageous to perform an up‐front enrichment of modified peptides prior to MS analysis. The most successful strategies for achieving this enrichment have been the use of affinity‐ or antibody‐based methods. Although these quantative proteomics approaches have been very successful in identifying thousands of regulatory PTMs in a single experiment, they are highly biased towards detection of specific modification. In order to achieve a thorough understanding of cellular signaling, identification of all components in the entire signaling network is required, including all proteins and PTMs. And little attention has been paid towards the presence of other modifications regulating intracellular signaling. Many key modifications involved in signaling processes have only been discovered in recent years such as methylation of arginines, ubiquitination, SUMO‐ylation, acetylation and O‐GlcNAc glycosylation. As a result, an unbiased platform to identify the full extent and functional importance of all protein modifications involved in signal transduction still needs to be realized.
We will investigate the biological role of novel modifications identified by the group using peptide pulldowns. Bait-fishing combined with quantative SILAC proteomics will elucidate interaction partners to the modification of interest using peptide sequences containing the investigated modification. These modified peptide sequences will be generated through standard Fmoc peptide synthesis. Additionally, we will perform proteome-wide analysis of novel PTMs by raising highly specific antibodies and/or by developing enrichment strategies that targets the modification of interest. This ensures that large scale mapping of specific modifications can be achieved during e.g. different types of cell stimuli or gene knock‐out experiments using quantative SILAC experiments. In collaboration with the other groups at the protein center we will carry out biological validation, apply the platforms to disease biology as well as perform bioinformatic analysis of our discoveries.
Furthermore, a novel proteomics platform will be developed for comprehensive mapping of signaling partners to secondary messengers such as phosphatidylinositol phosphates and other phospholipids. We will work in close collaboration with the recombinant protein production facility at CPR for generation and validation of specific protein domains identified as interaction partners.
The technology and platforms developed in the group will, when combined with the skills and expertise in the other departmental groups, allow for an unprecedented and comprehensive analysis of virtually all constituents in any cell signaling network.
Chunaram Choudhary, PhD: Proteomics and Cell Signaling
Our group aims to study cell signaling events, especially with relevance to human health and diseases, on a global level. Towards this goal, we will employ state-of-the-art proteomics technologies, in conjunction with cell and molecular biology techniques. We will work in close collaboration with the other groups within the proteomics department as well as with the other departments at the Centre, thereby benefitting from their expertise in the area of bioinformatics, systems biology, recombinant protein production, and disease biology.
Biological systems respond to extracellular and intracellular clues (signals) in myriad ways. Exposure of eukaryotic cells to extracellular growth factors and cytokines regulate many biological processes such as cell division, proliferation, growth, migration, and death. Cell proliferation and division are also controlled by intracellular signaling such as DNA damage response. Cells respond to these signals by modulating protein expression and their posttranslational modifications (PTMs). PTMs such as phosphorylation, acetylation, and ubiquitylation, play critical role in controlling nearly all cellular processes. Deregulation of protein expression and PTMs are associated with many human diseases such as cancer and neurodegenerative disorders.
Until recently, use of modification-specific antibodies was the major tool to study cell signaling. However, this approach has severe limitations for unbiased and high throughput analysis of cell signaling events on a global scale. Recent advances in quantitative proteomics have opened new ways for unbiased analysis of proteomes and PTMs on a global scale. Combining high-resolution mass spectrometry with SILAC offers unparalleled opportunities for quantitation of changes in protein expression and PTMs. We plan to employ this approach to quantify expression of thousands of proteins and tens of thousands PTMs in a single experiment. We will apply this powerful approach to study cell signaling on a ‘systems-wide' format to discover signaling pathways relevant to human health and diseases such as cancer. To this effect, we plan to quantify proteome and phosphoproteome of mammalian cells upon different perturbations. Functional roles of selected candidates from these screens will be examined using a variety of cell and molecular biology techniques. Furthermore, to understand the molecular mechanisms of these proteins and to indentify protein complexes, we will perform full-length protein pull-downs. Also, to indentify protein interactors that specifically bind to posttranslationaly modified versions of proteins; we will perform peptide pull-downs using modified or unmodified synthetic peptides.
The results from these global cell signaling studies and sophisticated downstream bioinformatics analysis are expected to provide a `systems-wide` view of signaling networks which will help to expand our understanding of regulatory mechanisms of cellular functions.