Protein adducts at critical protein sites as markers of toxicological risk

Getty, Paul (2014) Protein adducts at critical protein sites as markers of toxicological risk. PhD thesis, University of Glasgow.

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The formation of conjugates between the electrophilic reactive metabolites of drugs and nucleophilic protein sites is known to be associated with toxicological risk. At present there is no low cost and high throughput means of reliably detecting the presence of drug-protein adducts in vitro or in vivo. The development of a reliable high throughput methodology would facilitate the study of underlying mechanisms of toxicity and prove useful in early screening of potential drug molecules. Assays using liver microsomes and trapping agents such as glutathione are used to produce and detect a wide range of drug reactive metabolites which are then characterised by mass spectrometry. The glutathione trapping is effective for metabolite identifications but, the modification of proteins by means of electrophilic attack on nucleophilic centres often occurs in an enzyme independent manner and is unlikely to be analogous to the glutathione model. In order to create a more suitable model system, three short polypeptides were designed and synthesised. These peptides were incubated with clozapine and human liver microsomes. The resulting metabolite-peptide conjugates were analysed by nanoLC-MS. Results indicated that a characteristic conjugate specific ion at 359.1 Da could be detected for each of the peptides. This data was used to create a precursor ion scan specific for the presence of this characteristic ion.
Protein separation techniques including SCX, Offgel IEF and 1d-gel electrophoresis, in conjunction with LC-MS (with the precursor 359 scan), were applied to microsome prep samples in order to identify modified proteins. Using these approaches some 1700 protein identifications were made, more than 1000 of these were unique hits. The precursor ion scan was found to have poor selectivity identifying roughly 1/3 as many proteins as the information dependant acquisition approach. No drug-protein adducts were identified.
Further to this a novel application of saturation DIGE was applied in order to enrich for the presence of protein adducts. The DiGE approach was used to identify some 15 proteins with apparent change in abundance (fluorescence intensity) between clozapine treated and untreated samples. Spots were excised from the 2d gel digested and analysed by reversed phase liquid chromatography mass spectrometry. The IDA scans identified some 147 unique protein hits, the precursor ion scans identified 18. Again no drug-protein adducts were found.
Biotinylated desmethyl clozapine was metabolised in the human liver microsome assay. Western blotting was carried out on a 2d gel run from an assay sample. The Western membrane was probed using an HRP-Streptavidin probe. Imaging of the membrane revealed the presence of several biotin bearing proteins, many of which were not present in the negative control sample. A print out of the image was used as a map for the excision of modified proteins from a duplicate gel. Digestion and LCMS analysis of the samples revealed the presence of several proteins but no protein-adducts were found.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: protein adducts, biomarker, drug trapping, protein separation, proteomics
Subjects: Q Science > Q Science (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Andrew, Prof. Pitt
Date of Award: 2014
Depositing User: Mr Paul R Getty
Unique ID: glathesis:2014-4886
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 Apr 2014 10:28
Last Modified: 10 Apr 2014 10:30

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