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Title: Development of a method for the detection and quantification of the reaction products formed between aldehydes and proteins using high resolution mass spectrometry
Author: Ahmed, Ahmed S.
Awarding Body: University of Strathclyde
Current Institution: University of Strathclyde
Date of Award: 2010
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Detection of endogenous biomarkers is essential for the identification of certain abnormalities within biological systems which may result from different physiological or pathological conditions. Abnormalities within a biological system can result in excessive lipid peroxidation which can lead to the formation of certain lipo-peroxidation by-products, such as 2-alkenals, 4-hydroxy-2-alkenals and malondialdehyde (MDA). Such endogenous aldehydes can induce protein modifications and the formation of a set of biomarkers known as advanced lipo-peroxidation end products (ALEs). Previous detection methods which used for the detection of the modified proteins include immunohistochemical analysis, western blot analysis, 2D-gel electrophoresis, GC-MS, LC-MS, UV-visible and fluorescence detection. Most of these methods require pre-purification and off-line extraction and derivatisation steps prior to analysis [3]. Proteomics and immuno-assays have been used as a diagnostic tool for such biomarkers; however these techniques are not specific. In the case of immunohistochemical methods, cross reactivity can result in a non-specific signal. With proteomics approaches it is impossible to control the fragmentation processes for the protein molecule and multi-stage fragmentation is required to predict the site of modification. In this research project two different methods were developed for the qualitative and quantitative analysis of biomarkers resulting from the modification of proteins by aldehydes. The first method involved the analysis of amino acids within serum albumin (human or bovine) which had been modified by reaction with a series of different 2-alkenals or 4-hydroxy-2-nonenal (HNE). The protein sample was reacted with these aldehydes and the reaction products were subjected to reductive stabilisation with NaBH4, and were then hydrolysed with 6N HCl for 4 hrs at 145°C. The hydrolysis products were extracted and derivatised with propylchloroformate and then analysed by reversed phase liquid chromatography-mass spectrometry (RPLC-MS). The derivatisation method for the analysis of the reaction products between endogenous aldehydes (2-alkenal and HNE) and proteins was successful, but was complicated by the fact that partial derivatisation was possible. The second method of analysis was based on the recently developed separation technology of hydrophilic interaction liquid chromatography (HILIC) which enabled retention of the aldehyde adducts, without the need for a derivatisation step. This method involved the same initial procedure as the first one, with the exception that the hydrolysis products were extracted using a protein crash plate filter (PPT filter) and then analysed by HILIC in combination with Fourier Transform mass spectrometry (HILIC-FTMS). The retention of the un-derivatised adducts on the HILIC column was efficient, and a combination of high organic solvent content in the chromatographic mobile phase and a free positively charged amine group in these adducts allowed sensitive detection. High resolution mass spectrometry using an LTQ-Orbitrap instrument was able to characterise a wide range of aldehyde adducts with high sensitivity and minimum deviation of the observed masses from those of the theoretical masses. The fragmentation pattern of these adducts obtained using collision induced dissociation (CID) to enable structure elucidation for these compounds. Finally, limit of detection (LOD) and limit of quantification (LOQ) for amino acids and the related aldehyde adducts was determined. The aim of this project was to implement a mass spectrometry detection protocol for these biomarkers in hospitals and other clinical research centres. Examination of human plasma from normal subjects revealed that a number of 2-alkenal adducts were present.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available