It is widely reported that during periods of inflammation the heme enzyme, myeloperoxidase is generated by macrophages producing reactive oxidative species. Oxidative stress is the imbalance of these oxidative species which will lead to the post translational modification of proteins. Some biomarkers are proteins or post translational modifications that can be used to indicate disease and are becoming increasingly important particularly for the study of progressive diseases. Analysis of biomarkers in bodily fluids will not only be faster and less invasive than a biopsy but will also diagnose disease at an earlier stage and allow disease treatment and progression to be monitored. Known biomarkers for the production of myeloperoxidase are chlorotyrosine and nitrotyrosine. Elevated levels of chlorotyrosine and nitrotyrosine are indicative of atherosclerosis. The early diagnosis of atherosclerosis is important as the onset of this disease can occur at a young age and be asymptomatic until later, more developed stages. Here I aim to develop sensitive methods of detection for these biomarkers in a hope that they can be used to classify disease. A Qtrap mass spectrometer is employed with precursor scan for the selective and sensitive detection of chlorotyrosine modifications in in vitro HOCl modified 9 protein mix samples. Compared to a conventional MSMS experiment the precursor scan detects more chlorotyrosine modifications suggesting it is a better method for the detection of post translational modifications. Additionally the precursor scan can be used when there is no prior knowledge of the modification sites. A multiple reaction monitoring method was developed from the MSMS analysis of in vitro chemical modification of human serum albumin and plasma samples. Observations from the MSMS analysis were employed to write the multiple reaction monitoring method to target for chloro- and nitrotyrosine modified peptides of the human serum albumin protein in plasma samples. Detection of these modified peptides was indicated by the common elution of three transitions specific to the peptides precursor mass. Where anomalous peaks of one transition were seen it was known that this was not the elution of the targeted peptide. The use of three transition masses instead of one reduces the generation of false positives. Where more than one peak for the common elution time was seen for a targeted peptide in the chromatography gradient the retention times were used for identification. Peptides are separated by liquid chromatography prior to their analysis on the Qtrap by their hydrophobicity or their polarity. When a peptide becomes chloro- or nitrotyrosine modified the peptide becomes less polar and therefore is seen later in the gradient than in the unmodified state. The observation of more than one peak where the three transitions are seen to be commonly eluted was caused by break-through of signal from poor selection of a m/z value in Q1. The multiple reaction method developed from the analysis of in vitro chemically modified human serum albumin and plasma was then applied for the analysis of clinical samples in the hope that the chloro- and nitrotyrosine modified peptides targeted for in the samples could be used to classify disease. The clinical plasma samples were sourced from 12 healthy volunteers and 12 diseased cardiovascular patients. The multiple reaction monitoring method indicated the modification of peptides and the presence of these modified peptides was confirmed using targeted MSMS. Classification of these samples was not successful and it was thought that a combination of biomarkers is required for the classification of disease.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:529138 |
| Date | January 2011 |
| Creators | Thomson, Katrina |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/2408/ |
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