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Investigation of biological matrices for novel biomarkers by modern mass spectrometric methods. / Amy Ruth Godfrey

Swansea University Author: Amy Ruth Godfrey

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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...

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Published: 2008
Institution: Swansea University
Degree level: Doctoral
Degree name: Ph.D
URI: https://cronfa.swan.ac.uk/Record/cronfa42718
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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. 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spelling 2018-08-02T16:24:30.2270047 v2 42718 2018-08-02 Investigation of biological matrices for novel biomarkers by modern mass spectrometric methods. 89cb35a3547bf7616e7a4bf753d47c4e NULL Amy Ruth Godfrey Amy Ruth Godfrey true true 2018-08-02 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. E-Thesis Bioengineering.;Analytical chemistry. 31 12 2008 2008-12-31 COLLEGE NANME Biosciences COLLEGE CODE Swansea University Doctoral Ph.D 2018-08-02T16:24:30.2270047 2018-08-02T16:24:30.2270047 Faculty of Science and Engineering School of Biosciences, Geography and Physics - Biosciences Amy Ruth Godfrey NULL 1 0042718-02082018162516.pdf 10807487.pdf 2018-08-02T16:25:16.5600000 Output 25017185 application/pdf E-Thesis true 2018-08-02T16:25:16.5600000 false
title Investigation of biological matrices for novel biomarkers by modern mass spectrometric methods.
spellingShingle Investigation of biological matrices for novel biomarkers by modern mass spectrometric methods.
Amy Ruth Godfrey
title_short Investigation of biological matrices for novel biomarkers by modern mass spectrometric methods.
title_full Investigation of biological matrices for novel biomarkers by modern mass spectrometric methods.
title_fullStr Investigation of biological matrices for novel biomarkers by modern mass spectrometric methods.
title_full_unstemmed Investigation of biological matrices for novel biomarkers by modern mass spectrometric methods.
title_sort Investigation of biological matrices for novel biomarkers by modern mass spectrometric methods.
author_id_str_mv 89cb35a3547bf7616e7a4bf753d47c4e
author_id_fullname_str_mv 89cb35a3547bf7616e7a4bf753d47c4e_***_Amy Ruth Godfrey
author Amy Ruth Godfrey
author2 Amy Ruth Godfrey
format E-Thesis
publishDate 2008
institution Swansea University
college_str Faculty of Science and Engineering
hierarchytype
hierarchy_top_id facultyofscienceandengineering
hierarchy_top_title Faculty of Science and Engineering
hierarchy_parent_id facultyofscienceandengineering
hierarchy_parent_title Faculty of Science and Engineering
department_str School of Biosciences, Geography and Physics - Biosciences{{{_:::_}}}Faculty of Science and Engineering{{{_:::_}}}School of Biosciences, Geography and Physics - Biosciences
document_store_str 1
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description 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.
published_date 2008-12-31T03:53:31Z
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score 11.037253

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