Please use this identifier to cite or link to this item: http://hdl.handle.net/2445/171798
Title: Nuevas estrategias para la mejora de la sensibilidad y la selectividad en el análisis de biomarcadores proteómicos y miRNómicos en fluidos biológicos mediante CE-MS
Author: Peró Gascón, Roger
Director/Tutor: Sanz Nebot, María Victoria
Benavente Moreno, Fernando J. (Julián)
Keywords: Espectrometria de masses
Electroforesi capil·lar
Proteïnes
Àcids nucleics
Mass spectrometry
Capillary electrophoresis
Proteins
Nucleic acids
Fluids
Issue Date: 3-Nov-2020
Publisher: Universitat de Barcelona
Abstract: [spa] En las últimas décadas, el análisis de compuestos biomarcadores en muestras biológicas se ha convertido en una herramienta esencial para el diagnóstico, seguimiento y pronóstico de diversas enfermedades. Las principales dificultades en muchos casos son la baja concentración de los biomarcadores, la complejidad de la matriz de la muestra y la limitada disponibilidad de muestra. En esta tesis doctoral, se presentan nuevas estrategias basadas en electroforesis capilar- espectrometría de masas (CE-MS) para la separación, detección, caracterización y cuantificación de péptidos, proteínas y microARNs (miRNAs) biomarcadores en fluidos biológicos. La CE-MS es una técnica excelente para la separación de biomoléculas cargadas y su identificación inequívoca. En esta tesis, se han desarrollado estrategias novedosas para la predicción y optimización de las separaciones de mezclas complejas de péptidos en CE. Para evaluar estas estrategias, se han estudiado los péptidos beta amiloides. Una limitación importante de la CE es su baja sensibilidad en términos de concentración para la mayoría de analitos a causa del pequeño volumen de inyección de muestra. La extracción en fase sólida en línea con la electroforesis capilar (SPE-CE) es una excelente estrategia para disminuir los límites de detección. En esta tesis se han investigado diferentes metodologías de SPE-CE-MS unidireccional empleando sorbentes selectivos, como los sorbentes de inmunoafinidad, afinidad a aptámero, afinidad a metal inmovilizado y el carburo de silicio, para el análisis de proteínas intactas (transtiretina y α-sinucleína), digestos de proteínas y miRNAs en fluidos biológicos. También se ha investigado el potencial de la SPE-CE-MS con una nanoválvula (nvSPE-CE-MS) para el análisis de péptidos en fluidos biológicos. Se ha desarrollado un método de nvSPE-CE-MS empleando un sorbente C18 para el análisis de péptidos opioides y péptidos beta amiloides y se han comparado las ventajas e inconvenientes de la nvSPE-CE-MS respecto a la SPE-CE-MS unidireccional. Finalmente, se han investigado nuevas estrategias para el análisis rápido y eficiente de digestos de proteínas. La tripsina es la enzima proteolítica más comúnmente utilizada y la digestión se realiza habitualmente en disolución, requiriéndose largos tiempos de digestión. Con el objetivo de disminuir el volumen de muestra, su manipulación y el tiempo total de análisis, en esta tesis se ha desarrollado una metodología de análisis de proteínas empleando microrreactores empaquetados con partículas con tripsina inmovilizada en línea con la CE-MS (IMER-CE-MS).
[eng] In recent years, an increased emphasis has been placed on the analysis of biomarker compounds in biological samples for the diagnosis, follow-up and prognosis of numerous diseases. The leading difficulties in many of these analyses are the low concentration of the biomarkers, their structural complexity, the sample matrix effects and the limited availability of sample. In this doctoral thesis, we present novel strategies based on capillary electrophoresis- mass spectrometry (CE-MS) for the separation, detection, characterization and quantification of peptide, protein and microRNA (miRNA) biomarkers in biological fluids. MS is a powerful technique for the unequivocal identification of biomolecules due to its potential with regard to the detailed structural characterization of unknown compounds. However, due to the complexity of most biological fluids, the hyphenation of high-performance separation techniques is essential prior to MS analysis. In this regard, CE is a microscale technique that is very suitable for the separation of charged biomolecules. Nevertheless, electrophoretic separation methods must be properly optimized to achieve rapid, efficient, sensitive and high-resolution separations. In this thesis, we have investigated novel strategies to predict and optimize the separation of complex mixtures of peptides in CE. To assess these strategies, amyloid beta peptides, which are biomarkers of Alzheimer’s disease, were studied. A major limitation of CE is the relatively poor concentration sensitivity for most analytes due to the small sample volume that can be injected in the separation capillary. On-line solid- phase extraction capillary electrophoresis (SPE-CE) is a powerful approach for sample clean-up and reduction of the limits of detection. In SPE-CE, analytes from a large volume of sample are retained on a sorbent contained in a microcartridge. In unidirectional SPE-CE, the most typical configuration, the microcartridge is mounted in series to the separation capillary, inserted near the capillary inlet. After sample loading, the microcartridge is washed to remove non-selectively retained molecules. Then, the retained analytes are desorbed in a small volume of eluent, resulting in sample clean-up and concentration enhancement before electrophoretic separation and detection. In the present thesis, selective sorbents, such as immunoaffinity, aptamer affinity, immobilized metal affinity and silicon carbide sorbents have been explored in unidirectional SPE-CE-MS for the analysis of intact proteins, such as transthyretin, which is a biomarker of familial amyloidotic polyneuropathy type I, and α-synuclein, which is related to Parkinson’s disease, protein digests and miRNAs, which are related to cancer, in biological fluids. Unidirectional SPE-CE-MS is straightforward to implement. However, this simple setup has some inherent limitations. On the one hand, the sample volumes introduced using pressure depend on the dimensions of the separation capillary. On the other hand, sample loading is conducted in the same direction as the subsequent separation. Therefore, some of the matrix components could be irreversibly adsorbed in the inner wall of the separation capillary. Furthermore, in many cases, the requirements of on-line preconcentration are incompatible with the BGE necessary for an efficient separation or sensitive MS detection. In order to overcome these drawbacks, some new configurations where the sample is introduced in an orthogonal direction to the separation have been proposed, requiring the use of valves. We have investigated SPE-CE-MS with a nanoliter valve (nvSPE-CE-MS). A nvSPE-CE-MS method with a C18 sorbent for the analysis of opioid peptides and amyloid beta peptide fragments has been developed and the advantages and disadvantages compared to the unidirectional SPE-CE- MS have been discussed. New strategies for high-throughput bottom-up analysis of proteins have also been investigated. Tryptic digestion has been traditionally conducted in solution and requires long digestion times. In contrast, immobilized enzymes allow decreasing the sample volume and the total digestion times, minimize the sample handling, improve the digestion yields, as well as to stabilize the enzyme, avoid its autoproteolysis and simplify its recovery making it reusable. In this thesis, we have investigated on-line immobilized enzyme microreactor capillary electrophoresis-mass spectrometry (IMER-CE-MS) using microreactors packed with immobilized trypsin particles.
URI: http://hdl.handle.net/2445/171798
Appears in Collections:Tesis Doctorals - Departament - Enginyeria Química i Química Analítica

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