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Title: Investigation of biological matrices for novel biomarkers by modern mass spectrometric methods
Author: Godfrey, Amy Ruth
Awarding Body: Swansea University
Current Institution: Swansea University
Date of Award: 2008
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The primary objective was to introduce novel or develop existing techniques for the identification of new biomarkers within a range of biological matrices by modem mass spectrometric methods. Samples interrogated were hemodialysis concentrate, whole tissue sections and whole blood, with each having inherent challenges for use with mass spectrometry. Hence, published research has focused on other biological matrices or modes of detection for achieving the relevant aim. This current work overcame these issues by improving sample preparation including, the use of existing protocols for completely novel applications. Haemodialysate solution has proved most fruitful for identifying new candidate biomarkers. We have reproducibly detected 15 known and 6 novel uremic solutes within hemodialysate, a biological matrix previously deemed unsuitable for liquid chromatography/electrospray ionisation-mass spectrometry (LC/ESI-MS). This work included a validation of the novel methodology with stability and reproducibility investigations to test robustness. This highlighted a previously unrecorded thermally labile nature of some uremic solutes within the dialysate solution. A putative structural assignment has been made for 4 novel uremic solutes named, 5-(amino-1,2,-dihydroxy-ethyl)-3-nitrosooxy-[ 1,2,4]trioxine-3,6-diol, 2-(5,6-diamino-6-diazenyl-cyclohex-l-enyl)-2-hydroxy-acetimidic acid, N-[2-(7-hydroxy-3-methyl-ocatahydro-imidazo[ 1,5-alpha]pyridine-6-yl)-2-oxo-acetyl]-guanidine, and 3-(6-hydroxy-cyclohexa- 1,3-dienyl)-2-imino-3-oxopropionaldehyde. We have also identified that the chemical nature of solutes will dictate their removal during dialysis treatment and highly polar conventional biomarkers, urea and creatinine, are not representative of non-polar analyte excretion. This allows us to knowledgably suggest recommendations to improve future treatment modalities. The mass spectrometric analysis of whole tissue sections, in particular those that are paraffin embedded, pose a new range of challenges. Current MALDI matrices are unable to penetrate deep within tissue limiting their use to the tissue surface only. We have evaluated a range of novel dansylated MALDI matrices for this purpose that is detectable by fluorescence spectroscopy to aid in locating the matrix compound following application. Each dansylated MALDI matrix showed better penetration into the tissue sections, yet maintaining fluorescence detection, when compared to standard matrices CHCA, sinapinic acid and DHB. Of these novel matrices dansylhydrazine proved most successful in ionising proteins and peptides by forming a protonated molecule and related adducts. These additional mass shifted peaks, when included in a tryptic peptide database search, can improve the probability of the original protein/peptide identification. We now have the potential to obtain a total image of frozen tissue by using CHCA and dansylhydrazine in combination to ionise proteins/peptides at the surface or at depth, respectively. Further work is required for the preparation protocols with paraffin embedded sections for this total imaging principle to be applied. Finally we have illustrated the advantages of discovering novel haemoglobin variants in blood with a new ion mobility time-of-flight mass spectrometer, the Synapt HDMS system (Waters, MA, USA). We have identified a new variant that co-elutes with glycated haemoglobin peaks present in chromatograms used for conventional blood screening. Ion mobility technology and data extraction enhances the clarity of the results regarding multiple charging and variant characteristics. This enabled the exact determination of the amino acid substitution or mutation for the variant, with its assignment to a haemoglobin chain and the specific location within the chain.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available