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

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.