Current projects
Protein Expression Analysis
Proteomic analysis of angiogenic signaling networks
Professor Harry Mellor.
School of Biochemistry, University of Bristol.
Angiogenesis is the process by which the body extends new blood vessels. It is triggered in tissues that are deprived of oxygen - for example during heart disease or stroke. This formation of new blood vessels increases blood supply to the damaged area and plays a critical role in healing and repair. The signals produced by damaged tissue are detected by receptors on the blood vessel cells called VEGFRs. These interact with internal components of the cells to mobilize them for blood vessel outgrowth. We have constructed cell lines containing tagged versions of these receptors, allowing us to purify large amounts of receptor complexes from these cells. We are using the Proteomics Facility to analyze these complexes to discover the intracellular components that interact with the receptors and then drive angiogenesis. This is allowing us to map the complex networks of signals that control the process of angiogenesis, and thereby to identify potential drug targets for the clinical manipulation of this important pathway.
Using LC-MS/MS proteomics to understand and predict antibiotic resistance in bacteria.
Dr Matthew Avison.
School of Cellular and Molecular Medicine, University of Bristol.
Antibiotic resistance is sometimes caused by mutation, often in complex regulatory networks that control antibiotic permeability through the envelope. We use whole cell and envelope-only proteome analysis to identify which proteins are differentially regulated in novel antibiotic resistant mutants – selected in the lab or from infections that have failed therapy – in order to understand how the novel resistance mechanism works. We also use proteomics to see what happens in a cell when the novel regulators of resistance we discover have been experimentally manipulated.
The main cause of antibiotic resistance is the acquisition of mobile resistance genes and we use whole cell proteome analysis to determine the physiological response to carriage of these resistance genes. Using parallel fitness and resistance expression studies we aim to understand why some resistance genes are very commonly carried by certain strains of some species whilst others are not. This might allow us to predict the future clinical success of newly identified mobile resistance genes, informing targeted health-care surveillance. In doing this work we have been struck by the remarkable sensitivity of Orbitrap LC-MS/MS analysis to identify resistance proteins in complex samples. Our future aim is to exploit this sensitivity to identify which resistance proteins are being produced by bacteria in samples from infected patients – predicting what antibiotic the patient’s infection might best respond to without needing to wait for lengthy culture and sensitivity tests.
Characterising the Class I PI3K signalling network in human platelets.
Dr. Ingeborg Hers and Dr Tom Durrant.
School of Physiology & Pharmacology, University of Bristol.
Platelets are anucleate cells that play an essential role in thrombosis and haemostasis. They express all four Class I Phosphoinositide 3-kinase (PI3K) isoforms, each of which can catalyse the formation of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3). The use of gene targeted mice and small molecule inhibitors has revealed the importance of the Class I PI3K isoforms in platelet function, but the mechanisms of Class I PI3K activation and the events downstream of subsequent PtdIns(3,4,5)P3 generation are, in many cases, poorly understood. Class I PI3Ks associate with receptors and/or other proteins to permit their activation, while the resulting PtdIns(3,4,5)P3 most commonly regulates cell function via the recruitment and localisation of a range of PtdIns(3,4,5)P3 -binding proteins. We utilise affinity purification approaches, including Class I PI3K subunit immunoprecipitation and PtdIns(3,4,5)P3 bead pull downs, coupled to LC-MS/MS analysis, to capture and identify Class I PI3K-interacting proteins and PtdIns(3,4,5)P3-binding proteins from human platelet lysates. A number of the proteins identified are under investigation in the lab, including the PtdIns(3,4,5)P3-binding proteins Bam32/DAPP1 and Rasa3, with our work demonstrating the latter to play an important role in the regulation of Rap1b and integrin αIIbβ3-mediated cell spreading. Thus, functional characterisation of platelet proteins identified by these proteomic approaches will permit a more thorough understanding of Class I PI3K biology in this important cell type.
SILAC
Proteomic analysis of endosomal sorting.
Professor Pete Cullen.
School of Biochemistry, University of Bristol.
In regulating the localization and activity of transmembrane cargo proteins and their associated components, the endosomal network regulates and fine-tunes numerous cellular processes. We seek to achieve a thorough understanding of the organisation and function of this network and how this contributes to human health and disease. Quantitative proteomic approaches are allowing us to address two key questions: the identification and characterization of the molecular machines that regulate cargo sorting; and the global, unbiased identification of the cargo proteins that each machine sorts [18,21]. We now wish to extend these approaches, first to examine the role of retromer, an ancient endosomal sorting complex, in neuroprotection in Parkinson’s disease [15] and secondly, by developing proximity-based proteomics to define the functional organization of the network.
Mass-spectrometry based identification of norovirus-host interactions.
Prof. Ian Goodfellow and Dr Edward Emmott.
Division of Virology, Department of Pathology, University of Cambridge, Cambridge, UK
Norovirus infections represent a major economic burden in developed countries, and cause upwards of 200,000 deaths annually. Viruses are obligate intracellular parasites that require the use of host cell machinery in order to replicate. Previous work from our group has identified that a norovirus protein (VPg), covalently linked to the virus genome is essential for translation of viral proteins and functions as a 5’ cap substitute, allowing interaction with the host translational machinery. In this project we aim to identify both modifications to the host translational apparatus in response to norovirus infection, as well as identifying host proteins interacting with VPg (or other viral proteins) from across the caliciviridae family.
To date, the use of SILAC-based quantitative proteomics for this project, with analysis performed by the University of Bristol Proteomics Facility, has allowed us to successfully identify modifications to the eIF complex upon virus infection, cellular interaction partners of individual eIF proteins (eIF4AI, and eIF4AII), and the importance of particular VPg point mutations on host cell interactions.
TMT
Generation of red blood cells in vitro from stem cell sources.
Dr Jan Frayne.
School of Biochemistry, University of Bristol.
The generation of human red blood cells (RBCs) in vitro for transfusion purposes is a major goal of health services globally to help meet therapeutic needs. In recent years advances in the development of systems for the generation of erythrocytes in vitro have progressed rapidly using progenitor cells isolated from adult peripheral blood, umbilical cord blood and human pluripotent stem cells. Such cells can be induced to differentiate efficiently down the erythroid pathway, however detailed characterization and comprehensive analysis of the protein expression profile of such in vitro erythroid cells is required to determine how similar these cells actually are to normal erythroid cells. Furthermore, erythroid cells generated from pluripotent stem cells have differentiation and enucleation defects, the molecular basis for which are not understood. To investigate differences in the proteome, and to identify key deficiencies in pluripotent stem cell derived erythroblasts, we are utilizing multiplex Tandem Mass Tag (TMT) labelling and Mass Spec analysis, which enables direct quantitative comparison of the proteome between the different erythroid cell populations [24].
Proteomic profiling of lung injury following cardio-pulmonary bypass.
Dr Andrew Durham, Dr Emad Al Jaaly, Professor Gianni Angelini and Professor Ian Adcock.
National Heart and Lung Institute, Imperial College London.
Open-heart surgery is carried out on more than 35,000 UK patients each year with an all cause 30-day mortality of 3-4%. However, the numbers of patients experiencing respiratory complications is much higher and is rising due to the increasing number of high-risk patients having open-heart surgery. Complications after surgery place an enormous burden on hospital resources and are associated with increased NHS costs.
Conventional open-heart surgery requires the use of the heart-lung machine, also known as cardiopulmonary bypass (CPB), for 1-2 hours or more depending on the complexity of the operation. During this time the heart is stopped and most of the blood supply is diverted from the heart and lungs to allow the operation to proceed in a largely blood-free environment. Diverting the blood supply from the heart and lungs and artificially inducing lung collapse have been associated with heart and lung injury, inflammation, major complications, and increased costs.
Over the years effective techniques have been developed to protect the heart from this injury. These involve feeding the heart with an artificial intermittent supply of blood containing protective chemicals. In contrast, no methods for protecting the lungs have been developed; the lungs are still routinely left collapsed in the chest cavities without ventilation and with a poor blood supply for the entire duration of CPB.
The aim of this study is to investigate in a clinical setting a strategy for lung protection during cardiac surgery that involves ventilating the lungs at low frequency to avoid the harmful effects of prolonged collapse during CPB. We have carried out a randomised controlled clinical trial to compare the strategy of ventilating the lungs during CPB, with the current standard practice of collapsing the lungs during the surgery, focusing in particular on the effects of the two strategies on markers of inflammation in the lungs and peripheral blood.
As part of this study we are using Tandem Mass Tagging (TMT) in order to investigate global changes to the amounts of proteins in the lungs of patients before and after bypass surgery. Using this technique we hope to better understand and therefore prevent lung injury during bypass surgery.
Translational Proteomics to Investigate Links between Hypertension and Dementia.
Professor Patrick Kehoe.
School of Clinical Sciences, University of Bristol.
Our main research interest is focused on understanding the mechanisms which underlie the long acknowledged, but still poorly understood, association between hypertension and blood pressure regulation in general and the development of Alzheimer’s disease (AD) and other dementias. In a pilot study, we used TMT methodology to measure the effect of hypertension on changes in the levels of proteins in post-mortem brain tissue from healthy controls and AD patients. This work has generated a number of molecular 'leads' to be validated. It is now the plan to succeed this work with at least two lines of investigation. First, we hope to manipulate the targets identified in the pilot study in rodent models, which would be mirrored by similar manipulations in vitro in a combination of cell culture, iPS or primary cell lines. The intention would also be to couple our original TMT studies of human brain tissue to additional human biosamples, predominantly plasma/serum and CSF. We hope that this will help translate the potential for the identification of new biomarkers to peripheral bodily fluids for earlier diagnosis of dementia, which remains of vital importance at present if we are ever to be able to develop some effective therapies to treat this disease.
Phosphoproteomics
Cardiac Protein Expression and Phosphorylation in Health and Disease.
Professor Saadeh Suleiman. School of Clinical Sciences, University of Bristol.
Part of our research is to study cardiac remodelling triggered by disease/stress in experimental models and in patients with heart disease. These include models of high-fat diet and coronary artery disease, adult patients undergoing open heart surgery for valve or coronary artery disease and paediatric patients with congenital heart disease. In an on-going project, the Proteomics Facility has developed a sophisticated analytical approach which combines Tandem Mass Tagging (TMT) for protein quantitation with phospho-peptide enrichment to enable us to monitor changes in both cardiac protein expression and phosphorylation during cardiac insults including cardioplegic ischaemic arrest [26]. Key proteins identified so far are those associated with oxidative stress, calcium cycling, inflammation, metabolism and survival/death signalling. The information obtained from these studies will be used in the design of cardio-protective strategies.
Using Phosphoproteomics to understand Eph regulated signalling pathways.
Professor Catherine Nobes and Miss Jessica Campbell.
School of Biochemistry, University of Bristol.
Eph receptors are tyrosine kinase receptors which, when activated by their ephrin ligand, lead to alterations in the actin cytoskeleton and thus changes in cell migration. Our research project aims to investigate the downstream signalling components that are regulated upon Eph receptor activation.
In cells grown in SILAC media, we have stimulated the EphB4 receptor using an ephrin-B2 ligand. Protein extracts from these cells were then digested and the peptides subjected to phospho-peptide enrichment using a TiO2-based approach. The resulting phospho-enriched sample was then analysed by nano-LC MSMS. This has led to the identification of several thousand phosphorylated proteins, of which approximately 200 show a change in their phosphorylation state in response to ephrin stimulation. By comparing multiple experiments where we have combined Eph stimulation with siRNA treatment, we have been able to build up a picture of Eph dependent signalling events.