Development of Advanced Mass Spectrometric Approaches for the Analysis of the Proteome of Serum from Women with Pre-Eclampsia

Lead Research Organisation: Keele University
Department Name: Inst for Science and Tech in Medicine

Abstract

Proteins are the biological machinery of the cell. When this machinery is altered in some way, for instance via modification of a component, addition or removal of one of the units which form this molecular factory, this changes the way in which the cell performs. Such changes are at the root of many diseases. One disease state where changes in amounts and/ or nature of proteins have been implicated is the pregnancy condition pre-eclampsia. In this disease, a poor blood supply to the placenta induces release of factors, at least some of which are proteins, into the mothers' bloodstream. These proteins damage the lining of walls of maternal blood vessels, causing both small and large blood vessels to become leaky. Increased vessel leakiness leads to fluid escaping from vessels, and thus the symptoms of pre-eclampsia, such as high blood pressure, increased protein content in urine, and swelling of the hands and face. Release of these factors from the placenta occurs before any observable clinical symptoms, and currently no screening methods exist. If we can identify these proteins from blood plasma, we can implement screening tests for these within standard prenatal care regimes. Biological fluids such as plasma contain a huge number of different proteins, all of which are present at levels which vary between individuals. More significantly, the levels of different proteins are very different from one another. For instance, albumin is present at very high levels (tens of milligrams per millilitre of plasma (10E-3g/ml) ), whilst interleukin 6, a molecule involved in cell growth, is present at very low levels (just picograms per millilitre (10E-12g/ml)). Methods to identify the nature of proteins within such samples using analytical chemical methods are termed proteomics. The first step in a proteomic workflow is to digest proteins into small pieces, called peptides, using specific enzymes such as trypsin from cow pancreatic extracts. This step converts a mixture of many thousands of proteins into a mixture of many hundreds of thousands of peptides. Peptides are then analysed by spraying them in a stream of solvent under electrical charge into a mass spectrometer. Identity of the peptides is typically established by colliding the charged peptides (ions) with inert gas, which causes them to fall apart in a predictable manner, allowing us to 'read off' peptide sequence. These methods are best-suited to small peptides, as larger peptides are more flexible and do not fragment efficiently. Newer methods of sequencing peptide ions use electrons to induce fragmentation. These methods work best with longer polypeptides. This study will use proteolysis under different conditions, such as different enzymes and using chemicals to block enzyme cutting sites. Enzymatic cleavage will be simulated on a computer and optimal cleavage conditions selected for experimental substantiation. Once a method has been established, this will be used on plasma samples taken from women with pre-eclampsia to identify proteins whose abundance is altered between 'normal' plasma (pregnant, non-pre-eclamptic) and samples taken prior to and after development of disease. This will enable both increased understanding of the causes of pre-eclampsia and establishment of a panel of markers which can be carried forward for establishment of prognostic testing of plasma samples during pregnancy. Eventually, this will allow identification, streaming and appropriate management of women who would otherwise suffer pre-eclampsia. An eventual outcome will be identification of possible targets for drug intervention in this life-threatening illness.

Publications

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Hart S (2015) [82-OR] in Pregnancy Hypertension: An International Journal of Women's Cardiovascular Health

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Hart S (2015) [203-POS] in Pregnancy Hypertension: An International Journal of Women's Cardiovascular Health

Related Projects

Project Reference Relationship Related To Start End Award Value
EP/E043143/1 01/10/2007 01/11/2009 £335,916
EP/E043143/2 Transfer EP/E043143/1 02/11/2009 01/03/2011 £114,553
 
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Exploitation Route Development of new databases and tools for investigation of biological/medically-relevant samples
Sectors Agriculture, Food and Drink,Healthcare,Manufacturing, including Industrial Biotechology

 
Description I have used the findings to talk to members of the public about science and medicine.
First Year Of Impact 2014
Sector Education
Impact Types Cultural

 
Description New Investigator Research Grant (NIRG) Only Research Boards May/Jun 2016 Infections and Immunity Board
Amount £557,547 (GBP)
Funding ID MR/P011241/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 04/2017 
End 03/2020
 
Description Collaboration on Helen Price BBSome NIRG (listed under further funding) 
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Country United Kingdom 
Sector Academic/University 
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Collaborator Contribution We will work together to characterise proteins from the Leishmania BBSome
Impact None as yet as new grant. Collaboration is between parasitology and mass spectrometry, so is multidisciplinary.
Start Year 2017
 
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Department School of Geography, Geology and the Environment
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Sector Academic/University 
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Impact Data are under analysis at the moment. Cross-disciplinary project involving parasitology, molecular biology and mass spectrometry/analytical chemistry.
Start Year 2012
 
Description Pre-eclampsia 
Organisation University College Cork
Department Department of Pharmacology & Therapeutics
Country Ireland 
Sector Academic/University 
PI Contribution Analysis of proteins from urine samples taken from patients with pre-eclampsia and intrauterine growth restriction
Collaborator Contribution Provision of samples to study - research nurse and research assistant time to pull samples and provide database information.
Impact Paper in Int J Mass Spectrom, PhD student with collaborative project.
Start Year 2011
 
Description Waters nanoparticle study 
Organisation Waters Corporation
Department Micromass UK Ltd
Country United Kingdom 
Sector Private 
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Start Year 2014
 
Description Keele Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Keele Community Day is a dedicated opportunity for the local community to see what Keele is about, with research and teaching-themed events. Each year at least 5,000 visitors come onto campus to visit us. We have held a variety of events, including a 'Forensic Bear Hunt'.
Year(s) Of Engagement Activity 2013,2015
URL http://www.keele.ac.uk/keeleday/
 
Description School visit (Manchester) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
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Year(s) Of Engagement Activity 2010
 
Description Soapbox Science Bristol 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
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Year(s) Of Engagement Activity 2016