When treating patients with cancer, the ability to predict a patient’s response to treatment is an important tool to allow therapy to be tailored for best outcome. Therefore, the need exists for a test to forecast a patient’s response using a sample that is readily accessible, and provides an accurate reflection of a patient’s response to a disease or treatment. Profiling biological fluids, such as plasma or urine, has gained considerable interest in recent years. This is because these fluids are readily available and are expected to provide an accurate representation of a patient’s response to treatment. As such, much effort has been put into finding biomarkers or prognostic indicators. Abnormal protease activity has been linked to the progression of cancer due to their involvement in several processes vital to the survival and proliferation of the disease. These include metastasis, resistance to apoptosis and angiogenesis, amongst others. In addition, dysregulated protease activity has been linked to poor response to chemotherapy as well as tumour regrowth following radiotherapy. Therefore, an increased understanding regarding the activity of a patient’s proteases may provide the clinician with more information as to how best to treat the patient. Therefore, monitoring protease activity has been suggested as a potential marker to predict a patient’s response to cancer treatment. Most enzyme activity assays are currently performed by fluorescence spectroscopy. However, these workflows suffer from limited sensitivity and linear range. Therefore, an alternative, more sensitive assay is required. Mass spectrometry (MS) is a highly sensitive analytical technique routinely used to quantify changes in biological systems. As such, MS has the potential to be used in enzyme activity assays. This study illustrates the development of a novel MS based method to monitor the activity of target enzymes in plasma; specifically asparaginyl endopeptidase (AEP) and caspase-3 using mass spectrometry. These enzymes have been linked to poor chemotherapeutic response in childhood leukaemia and tumour regrowth post-radiotherapy respectively. This project will describe the development and optimisation of each stage of a five step sample preparation and analysis method. This includes the design of enzyme substrates designed to be cleaved by the target enzyme, whilst reducing the effect of other enzymes acting on this substrate, how best to enrich samples for these target peptides, as well as determining the best MS technique to monitor these peptides. In addition, this project describes a comparison between this assay and an existing fluorescence assay when monitoring AEP activity in biological samples such as plasma and whole cell lysates. The application of this method in quantifying caspase-3 activity in plasma samples is also examined.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:606895 |
| Date | January 2012 |
| Creators | Potier, David N. |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://www.manchester.ac.uk/escholar/uk-ac-man-scw:185527 |
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