京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18824号 / 理博第4082号 / 新制||理||1587(附属図書館) / 31775 / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

Mass spectrometry coupled with liquid chromatography and gel electrophoresis enables separation and detection of components in a complex mixture. During the last two decades, its applications were dramatically extended and remarkable progress has been made in many fields, in particular, environmental and biological analyses. This dissertation focuses on identification and characterization of biologically active compounds and comparative analysis of protein expression changes. The first two projects (Chapters 2 and 3) focus on the application of LC/MS approach to profile the bioactivated intermediates of 4, 4'-methylenedianiline (DAPM) from rat vascular smooth muscle cells (VSMCs) and bile. In our study, several DAPM metabolites were detected and characterized in detail by liquid chromatography-electrospray tandem mass spectrometry. The structural assignments of these metabolites from VSMCs and rat bile significantly improve our understanding of DAPM biotransformations and toxicity. The third project described in Chapter 4 focuses on using electrospray tandem mass spectrometry (ES-MS/MS) and theoretical calculation (GAUSSIAN 03 program) to investigate the unusual methyl radical loss and consecutive fragment ions that dominate the low-energy collision induced dissociation (CID) mass spectra of prodiginine compounds. Structures of the fragment ions are proposed and explanations are given to rationalize the observed competition between the formation of even-electron ions and radical ions. Our study shows that the lower apparent threshold associated with methyl radical loss points to a lower kinetic barrier. In Chapter 5, hypoxia-induced changes of zebrafish skeletal muscle were studied using two-dimensional difference in-gel electrophoresis (2D-DIGE) in vivo after 48 h in hypoxia vs. normoxia. The results showed that proteins involved in mitochondrial oxidative metabolism are down-regulated, whereas glycolytic enzymes are up-regulated to compensate for the loss of ATP synthesis in aerobic metabolism. The up-regulation of two spots identified as hemoglobin variants was also observed. These protein expression changes are consistent with a hypoxic response that enhances anaerobic metabolism or O2 transport to tissues.

No description available.

The main focus of the PhD study was to develop new gel electrophoresis and ICP-MS based methods to analyze a wide variety of the bio-molecules such as proteins, phosphoproteins and metalloproteins etc. The tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (tricine-SDS-PAGE) method is commonly used to resolve low molecular mass proteins, however, it requires a high percentage gel and a very complicated procedure to achieve this separation. This study describes a modification to tricine-SDS-PAGE to make it more effective for the separation of smaller proteins and for coupling to ICP-MS. The modified method employs low percentage PAGE gels and low reagent concentrations that provide efficient separations, good quantitation and low matrix levels that are compatible with ICP-MS. This modified method was applied to analyze phosphopeptides. Phosphopeptides are very small in size and difficult to separate using the other techniques such as Laemmli SDS-PAGE, original tricine-SDS-PAGE, immobilized metal affinity chromatography (IMAC), size exclusion chromatography (SEC) etc. In this study a simplified procedure is described based on modifying the original tricine-SDS-PAGE method. A comparative study showed that this modified method successfully resolved a digest mixture of very low to high molecular mass phosphopeptides/peptides. In off-line coupling of this method with ICP-MS, much better recoveries of the peptides from the gel were obtained as compared to traditional methods which indicate the compatibility of this modified method for quantitative studies. An on-line coupling of the modified system with ICP-MS was also demonstrated and it was applied for the separation, detection and quantification of phosphopeptides. Another application of this modified system was the separation of serum proteins. Blood serum contains five major protein groups i.e., albumin, alpha-1 globulin, alpha-2 globulin, beta globulin and gamma globulin. The separation of these five major proteins in a single gel is difficult to achieve using traditional methods. The modified system was shown to be superior for the separation of these serum proteins in a 7% (m/v) native-PAGE gel and a cellulose acetate membrane. A further study was carried out into controlling the factors that cause metal loss and protein fragmentation in SDS-PAGE. Using a reducing sample buffer, and heating to high temperatures (90-100ºC) in alkaline or acidic conditions may cause protein fragmentation and decrease the metal binding affinity. 70ºC was found suitable to prepare the sample at neutral, alkaline or acidic pH as no fragmentation observed. To prevent metal loss, the binding constant (log K) values of metal-amino acids, play the major role. Those metals which have high binding affinities with the amino acids in proteins can also be affected by the variation of the pH so prior information about pH to maintain the binding constant values is essential to minimize metal loss. This was observed in the loss of zinc, and to a lesser extent copper from human serum albumin (HSA) as measured by inductively coupled plasma mass spectrometry (ICP-MS). The method described above was applied for the separation and quantification of the serum proteins obtained from age-related macular degeneration (AMD) patients (where the AMD patients were from Moorfields Eye Hospital, London). Zn and Cu were quantified employing external calibration. Zn concentration showed variation whilst Cu did not show any significant variations in samples from AMD patients. A brief study of the interaction of cisplatin and oxaliplatin with HSA and transferrin was also performed. Cisplatin bound much faster than oxaliplatin with HSA. After 24 hours incubation, cisplatin showed a decrease in signal intensity which indicates that cisplatin binding decreases with time. Cisplatin binding with transferrin as compared to HSA was not significant, which could be the result of unstable Pt-transferrin complex formation. Oxaliplatin did not show high binding to either protein, perhaps due to the presence of the bulky, non polar DACH ligand.

Fundulus is a diverse and widespread genus of small teleost fish of North America. Due to its high tolerance for physiochemical variation (e.g. temperature, oxygen, salinity), Fundulus is a model organism to study physiological and molecular adaptations to environmental stress. The thesis focuses on patterns of protein expression in Fundulus heteroclitus and F. grandis.The patterns of protein expression were investigated using traditional methods of enzyme activity measurements and recent proteomic approaches. The findings of the study can be used to guide future studies on the proteomic responses of vertebrates to environmental stress. Chapter 2 focuses on measurement of the temporal effects of oxygen treatments on the maximal specific activities of nine glycolytic enzymes in liver and skeletal muscle during chronic exposure (28d) of Fundulus heteroclitus. The fish was exposed to four different oxygen treatments: hyperoxia, normoxia, moderate hypoxia, and severe hypoxia. The time course of changes in maximal glycolytic enzyme specific activities was assessed at 0, 8, 14 and 28 d. The results demonstrate that chronic hypoxia alters the capacity for carbohydrate metabolism in F. heteroclitus, with the important observation that the responses are both tissue- and enzyme-specific. Chapter 3 studies the effect of tissue storage on protein profile of tissues of F. grandis. The technique of one dimensional gel electrophoresis (1D-SDS-PAGE) was used to assess the effects of tissue sampling, flash frozen in liquid nitrogen versus immersion of fresh tissue in RNA later, for five tissues, liver, skeletal muscle, brain, gill, and heart, followed by LC-MS/MS to identify protein bands that were differentially stabilized in gill and liver. The study shows that, in F. grandis, the preferred method of preservation was tissue specific. xi Chapter 4 focuses on the use of advanced 2DE-MS/MS to characterize the proteome of multiple tissues in F. grandis. Database searching resulted in the identification of 253 non-redundant proteins in five tissues: liver, muscle, brain, gill, and heart. Identifications include enzymes of energy metabolism, heat shock proteins, and structural proteins. The protein identification rate was approximately 50 % of the protein spots analyzed. This identification rate for a species without a sequenced genome demonstrates the utility of F. grandis as a model organism for environmental proteomic studies in vertebrates.

A avaliação das modificações no proteinograma do leite tem sido utilizada como meio de diagnóstico das mamites dos bovinos, pois, em decorrência dos processos inflamatórios na glândula mamária, ocorrem alterações, tanto na concentração dos componentes protéicos do leite, como também, surgimento de compostos protéicos não elaborados no processo de secreção láctea. A técnica de fracionamento protéico por eletroforese em gel de poliacrilamida mostrou-se eficiente na detecção de pequenas quantidades de proteínas em fluídos orgânicos. As características das proteínas do leite de vacas sadias, e a avaliação de possíveis fatores de variabilidade foram determinadas em 139 amostras de leite de vacas. Os animais foram, inicialmente, submetidos a exame clínico geral e do úbere, complementado por exames físico-químicos, celulares e microbiológicos do leite, complementares ao diagnóstico das mamites. Do conjunto das amostras, 97 eram de vacas sadias, em plena lactação, sendo utilizadas para estabelecer valores padrões dos constituintes do leite de vacas sadias e avaliar a influência racial - Jersey e Gir; e do número de lactações - animais em primeira lactação, duas ou três lactações e, quatro ou mais lactações sobre o proteinograma lácteo. Além do mais a amostragem serviu de base para a avaliação da fase da lactação sobre o quadro protéico e, permitiram a formação de grupos experimentais para avaliação da influência do número de células somáticas, do isolamento bacteriano e do estado de saúde do úbere no proteinograma lácteo, obtido por eletroforese em gel de poliacrilamida. Os resultados demonstraram influência significativa de fatores raciais no teor de proteína total do soro lácteo, e de suas frações imunoglobulínica, α1-antitripsina, β-lactoglobulina, bem como de um conjunto de proteínas do soro lácteo separadas, mas não identificadas; influência do número de lactações no teor de albumina de origem plasmática, imunoglobulinas, β-lactoglobulina e α-lactoalbumina. A fase da lactação influenciou de forma significativa os teores de proteína total do leite, assim como nas frações protéicas do soro lácteo de vacas sadias, variações evidentes na fase colostral da lactação. O número de células somáticas influenciou o proteinograma do leite, apenas nas amostras com mais de 1.500.000 células somáticas/ml. Não foi demonstrada a influência significativa do isolamento bacteriano no proteinograma lácteo de vacas. Entretanto, observou-se significativa influência da mamite no proteinograma do leite de vacas, agindo, principalmente, nas proteínas não sintetizadas no ciclo fisiológico de secreção da glândula mamária. / The evaluation of the modifications in milk proteinogram is used as a diagnostic tool for bovine mastitis, once the inflammation of the mammary glands leads to changes in the concentration of milk proteic components, as well as the appearance of proteic components that are not elaborated in the milk secretion process. The technique of protein fractioning by electrophoresis in polyacrylamide gel is effective in detecting small amounts of proteins in organic fluids. The features of milk proteins of healthy cows and the evaluation of possible variability factors were determined in 139 cow milk samples. Animals were first submitted to general clinical examination and udder examination, and to physical-chemical, cellular and microbiological milk tests, complementary to mastitis diagnosis. Of the total samples, 97 were from healthy cows in full lactation, used to establish reference values for the components of healthy cow milk and to evaluate the influence of breed - Jersey and Gir, and number of lactations - first, two or three lactations, and four or more lactations on milk proteinogram. Additionally, sampling was useful as a base for the formation of experimental groups for evaluation of the influence of the number of somatic cells, bacterial isolation and health condition of the udder on milk proteinogram obtained by electrophoresis in polyacrylamide gel. Results showed a significant influence of breed related factors on the level of total protein and its fractions immunoglobulinic, α1-antitripsin, β-lactoglobulin in whey, as well as on the level of a group of separated but not identified whey proteins; influence of the number of lactations on the level of albumin of plasmatic origin, immunoglobulins, β-lactoglobulin and α-lactoalbumin. The lactation phase influenced the levels of milk serum protein significantly, as well as the proteic fractions of healthy cow milk serum, variation evident in the colostrum phase of lactation. The number of somatic cells influenced milk proteinogram only in samples with more than 1,500,000 somatic cells. No significant influence of bacterial isolation on milk proteinogram was demonstrated. However, a significant influence of mastitis on the proteinogram of cow milk was observed, affecting mostly those proteins not synthetized in the physiologic cycle of mammary gland secretion.

Although inductively coupled plasma mass spectrometry (ICP-MS) is generally considered a reliable analytical technique, increasing demands on its capabilities require continued research and improvements. ICP-MS is susceptible to both matrix effects and drift, leading to a decline in accuracy and precision. A number of techniques are routinely used to compensate for these issues. Internal standardization is one such solution that requires relatively simple sample preparation and yet offers the possibility of improving both accuracy and precision. In order to be effective, an optimal analyte/internal standard pair must be chosen. Traditionally, analyte/internal standard pairs are chosen based on similarities in mass and/or ionization potential. The present studies sought to develop a program that determined standards based on the minimization of analytical error. 102 masses were monitored over 27 perturbations, i.e., changes to sample matrix and operating parameters. The standard deviations of the analyte/internal standard ratios were then used as a measure of internal standard performance. A thorough statistical analysis was conducted to determine trends between a good analyte/internal standard pair and similarities in chemical property. Similarities in mass offered the strongest relationship to a good internal standard choice, although many exceptions existed. The program was then tested over time and multiple instrument optimizations as well as on a completely different ICP-MS instrument. Results of these tests suggest that the data originally collected for the prediction program is not instrument-specific and thus provided a broader base of useful applications. Due to its unmatched sensitivity and multielement capabilities, ICP-MS is frequently utilized for biological samples. A more recent application, however, seeks to use ICPMS for the purpose of determining specific associations between metals and proteins. Such speciation requires a high resolution and reproducible separation prior to ICPMS analysis. Gel electrophoresis offers good separation and is well matched with the scanning properties of laser ablation sample introduction. The present study utilized native gel electrophoresis coupled with a uniquely modified electroblot system to improve sensitivity and to elucidate additional information. Chemically modified quartz fiber filters were successfully used as the transfer membrane to improve protein and metal capture efficiency. / text

A avaliação das modificações no proteinograma do leite tem sido utilizada como meio de diagnóstico das mamites dos bovinos, pois, em decorrência dos processos inflamatórios na glândula mamária, ocorrem alterações, tanto na concentração dos componentes protéicos do leite, como também, surgimento de compostos protéicos não elaborados no processo de secreção láctea. A técnica de fracionamento protéico por eletroforese em gel de poliacrilamida mostrou-se eficiente na detecção de pequenas quantidades de proteínas em fluídos orgânicos. As características das proteínas do leite de vacas sadias, e a avaliação de possíveis fatores de variabilidade foram determinadas em 139 amostras de leite de vacas. Os animais foram, inicialmente, submetidos a exame clínico geral e do úbere, complementado por exames físico-químicos, celulares e microbiológicos do leite, complementares ao diagnóstico das mamites. Do conjunto das amostras, 97 eram de vacas sadias, em plena lactação, sendo utilizadas para estabelecer valores padrões dos constituintes do leite de vacas sadias e avaliar a influência racial - Jersey e Gir; e do número de lactações - animais em primeira lactação, duas ou três lactações e, quatro ou mais lactações sobre o proteinograma lácteo. Além do mais a amostragem serviu de base para a avaliação da fase da lactação sobre o quadro protéico e, permitiram a formação de grupos experimentais para avaliação da influência do número de células somáticas, do isolamento bacteriano e do estado de saúde do úbere no proteinograma lácteo, obtido por eletroforese em gel de poliacrilamida. Os resultados demonstraram influência significativa de fatores raciais no teor de proteína total do soro lácteo, e de suas frações imunoglobulínica, α1-antitripsina, β-lactoglobulina, bem como de um conjunto de proteínas do soro lácteo separadas, mas não identificadas; influência do número de lactações no teor de albumina de origem plasmática, imunoglobulinas, β-lactoglobulina e α-lactoalbumina. A fase da lactação influenciou de forma significativa os teores de proteína total do leite, assim como nas frações protéicas do soro lácteo de vacas sadias, variações evidentes na fase colostral da lactação. O número de células somáticas influenciou o proteinograma do leite, apenas nas amostras com mais de 1.500.000 células somáticas/ml. Não foi demonstrada a influência significativa do isolamento bacteriano no proteinograma lácteo de vacas. Entretanto, observou-se significativa influência da mamite no proteinograma do leite de vacas, agindo, principalmente, nas proteínas não sintetizadas no ciclo fisiológico de secreção da glândula mamária. / The evaluation of the modifications in milk proteinogram is used as a diagnostic tool for bovine mastitis, once the inflammation of the mammary glands leads to changes in the concentration of milk proteic components, as well as the appearance of proteic components that are not elaborated in the milk secretion process. The technique of protein fractioning by electrophoresis in polyacrylamide gel is effective in detecting small amounts of proteins in organic fluids. The features of milk proteins of healthy cows and the evaluation of possible variability factors were determined in 139 cow milk samples. Animals were first submitted to general clinical examination and udder examination, and to physical-chemical, cellular and microbiological milk tests, complementary to mastitis diagnosis. Of the total samples, 97 were from healthy cows in full lactation, used to establish reference values for the components of healthy cow milk and to evaluate the influence of breed - Jersey and Gir, and number of lactations - first, two or three lactations, and four or more lactations on milk proteinogram. Additionally, sampling was useful as a base for the formation of experimental groups for evaluation of the influence of the number of somatic cells, bacterial isolation and health condition of the udder on milk proteinogram obtained by electrophoresis in polyacrylamide gel. Results showed a significant influence of breed related factors on the level of total protein and its fractions immunoglobulinic, α1-antitripsin, β-lactoglobulin in whey, as well as on the level of a group of separated but not identified whey proteins; influence of the number of lactations on the level of albumin of plasmatic origin, immunoglobulins, β-lactoglobulin and α-lactoalbumin. The lactation phase influenced the levels of milk serum protein significantly, as well as the proteic fractions of healthy cow milk serum, variation evident in the colostrum phase of lactation. The number of somatic cells influenced milk proteinogram only in samples with more than 1,500,000 somatic cells. No significant influence of bacterial isolation on milk proteinogram was demonstrated. However, a significant influence of mastitis on the proteinogram of cow milk was observed, affecting mostly those proteins not synthetized in the physiologic cycle of mammary gland secretion.

Peanut, a major allergenic food, has life-threatening potential and is difficult to be totally avoided due to its common use in processed foods. Thermal processing can influence the allergenic properties of peanuts. However, the kinetics of the reactions caused by thermal processing has not been characterized. In our study, kinetics of the commonly used thermal processing methods on a commercial peanut cultivar (Virginia) using five time intervals was conducted. Water-soluble and SDS-sample buffer soluble proteins were extracted sequentially, and analyzed by sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western-blot using human plasma containing IgE antibodies. The relationships between thermal processing (time) and log transformed water-soluble/total extractable major allergen content could be explained by a simple linear regression kinetic model for most of the processing methods (except high-pressure steaming). Among all the methods with optimal processing point, frying for 6 min had relatively lower IgE binding (linear epitopes) ratio may be due to the fact that this processing condition causing break down, cross-linking and aggregation of Ara h 2, and relatively lower solubility. Besides thermal processing, enzymatic processing also is considered to be an effective method in the allergenicity of peanuts. Eleven peanut lines (Coded MS-1~MS-11, MS-9 is the check and a common cultivar namely Valencia) were pre-screened from 122 peanut lines harvested in 2015 for allergen levels. These pre-screened lines were re-planted in 2016 for further analysis. One line, MS-7, was selected for lower Ara h 1 (8.5-9.5% of total protein) and Ara h 2 (4.2-6.6% of total protein) content in 2015 and 2016. Roasted MS-9 (check) peanut powders were used for enzymatic treatment for enzyme selection. A first order kinetic reaction model was conducted to describe the relationship between enzyme concentration (0-400AzU/g) and IgE-binding property reduction. Among eight food-grade enzymes, bromelain, papain and ficin hydrolysates had lower IgE-binding properties in terms of high IgE-binding property reducing rate (K, ≥ 0.4) and were selected for the following study. MS-7 (selected) & MS-9 (at level of 200AzU/g) hydrolyzed by three selected enzymes (200AzU/g) were used for IgE binding property comparison, TGase crosslinking and functional properties study. After hydrolyzed by the selected enzymes (200 AzU/g), the emulsion and foaming stabilities were decreased. Emulsion and foaming stabilities were increased in TGase (5U/g protein) crosslinked hydrolysates, which were even higher than soy protein isolate (SPI). The IgE-binding properties of TGase treated hydrolysates were similar to the hydrolysates without TGase treatment. MS-7 hydrolysates (with/without TGase) possessed less IgE-binding properties and similar functionality as compared with MS-9 hydrolysates.

Η μεσογειακή μύγα αποτελεί το κύριο παράσιτο πολλών καλλιεργούμενων φρούτων προκαλώντας ετησίως μεγάλες καταστροφές σε γεωργικές καλλιέργειες. Η μελέτη του εντόμου τόσο σε γενετικό όσο και σε πληθυσμιακό επίπεδο μπορεί να συμβάλλει σημαντικά στην ανάπτυξη ή και τη βελτίωση αποτελεσματικών και φιλικών προς το περιβάλλον μεθόδων ελέγχου. Οι μικροδορυφόροι είναι απλές επαναλήψεις ενός νουκλεοτιδικού μοτίβου που αποτελείται 1-6 ζεύγη βάσεων. Αποτελούν πολύ χρήσιμους γενετικούς δείκτες διότι είναι άφθονοι και διάσπαρτοι στο γονιδίωμα των ευκαρυωτκών οργανισμών. Επιπλέον είναι υψηλά πολυμορφικοί, κληρονομούνται ως συνυπερέχοντες Μεντελικοί δείκτες και αναλύονται εύκολα μέσω PCR, χαρακτηριστικά που τους καθιστούν πολύτιμα εργαλεία για πληθυσμιακές και εξελικτικές μελέτες. Από τους μικροδορυφορικούς δείκτες που αναπτύχθηκαν στο εργαστήριό μας, επιλέχθηκαν 10 με βάση το βαθμό πολυμορφισμού που έδειξαν σε εργαστηριακά στελέχη. Οι δείκτες αυτοί χρησιμοποιήθηκαν στην ανάλυση 481 ενηλίκων ατόμων που προέρχονταν από 19 διαφορετικά δείγματα φρούτων που συλλέχθηκαν από εννέα διαφορετικές περιοχές της Δυτικής Ελλάδας και της Βόρειας Πελλοπονήσου. Η γενοτυπική ανάλυση πραγματοποιήθηκε με ηλεκτροφόρηση των προϊόντων της PCR για κάθε γενετικό δείκτη σε πήκτωμα πολυακρυλαμιδίου και επακόλουθη αυτοραδιογραφία. Η στατιστική επεξεργασία των αποτελεσμάτων, με τη βοήθεια υπολογιστικών προγραμμάτων αποκάλυψε σημαντική γενετική διαφοροποίηση μεταξύ των δειγμάτων, που αποδίδεται εκτός των άλλων παραγόντων (κλιματολογικές συνθήκες, γεωγραφική προέλευση) στο είδος του ξενιστή. / C. capitata, is the main pest of many cultivable fruits and responsible for a significant loss in annual products, resulting in great economic damage. Studies on the genetic and population analysis will make a contribution towards the development or the improvement of environmental friendly control methods. Microsatellites are tandem simple sequence repeats of short (1-6) nucleotide motifs. They are very important genetic markers because they are dispersed and abundant in most eukaryotic genomes. They are highly polymorphic, inherited as co-dominant Mendelian markers and easily scored by PCR. Consequently, they have become one of the most popular molecular markers with application in many genetic studies, including genetic analysis of natural populations and evolutionary studies. From the available microsatellites in our laboratory, were selected ten (10), based on their polymorphism in laboratory strains. They were used for the screening of 481 adult flies in the medfly, collected from nineteen (19) different samples of fruits from nine (9) different areas in west Greece and north Peloponnesus. Analysis of genotype composition in the samples was achieved by polyacrylamide electrophoresis of the PCR products, for every genetic marker and then autoradiography. The statistic analysis of our results using software programs, revealed an important genetic differentiation in samples. Except for many factors (climatic conditions, geographic origin), the host origin will be responsible for this genetic differentiation.