Detección y caracterización de péptidos, proteínas y glicoproteínas como biomarcadores en fluidos biológicos. Aplicación al diagnóstico y seguimiento de diversas patologías

Abstract

Esta tesis doctoral se ha centrado en el desarrollo de metodologías analíticas adecuadas que permitan la separación, detección, caracterización e identificación de proteínas y metabolitos como biomarcadores de diferentes enfermedades o procesos patológicos. Concretamente, se ha centrado en el análisis de biomarcadores proteicos tales como la transtiretina (TTR), una proteína oligomérica con una gran tendencia a formar agregados amiloides y relacionada con la polineuropatía amiloidótica familiar tipo I (FAP-I), y el antígeno carcinoembrionario humano (CEA), una glicoproteína biomarcadora del cáncer de colon y de la metástasis de hígado (derivada del cáncer de colon). También se ha hecho especial énfasis en el estudio de biomarcadores metabólicos relacionados con la progresión de la enfermedad del Huntington, una enfermedad neurodegenerativa hereditaria caracterizada también por la formación de agregados amiloides.

La dificultad de las metodologías empleadas para el análisis de biomarcadores proteicos y metabólicos reside principalmente en el tratamiento de muestra que se tiene que llevar a cabo para aislar y preconcentrar los compuestos de interés presentes en muestras biológicas complejas (tales como suero y plasma) y en la utilización de técnicas de separación y detección adecuadas que permitan la correcta caracterización de dichos biomarcadores. Por este motivo, se han empleado diferentes estrategias para el análisis dirigido (targeted analysis) y no dirigido (untargeted analysis, global profiling) de biomarcadores proteicos y metabólicos, así como para el análisis de proteínas mediante estrategias de top-down y bottom-up, cada una con sus ventajas e inconvenientes. También se han evaluado diferentes técnicas de separación de alta resolución miniaturizadas como la electroforesis capilar (CE), la cromatografía de líquidos capilar (CapLC) y la movilidad iónica (IM), todas ellas acopladas a la espectrometría de masas (CE-MS, CapLC- MS y IM-MS, respectivamente). En resumen, esta tesis doctoral presenta una visión global de las estrategias analíticas más utilizadas actualmente para el estudio de biomarcadores en el campo de la investigación proteómica y metabolómica, con el objetivo de poder diagnosticar, predecir un debut temprano y proponer nuevas dianas terapéuticas a diferentes enfermedades y procesos patológicos.

The present thesis is focused on the separation, detection and characterization of proteins, glycoproteins and metabolites in biological fluids that may be able to act as disease biomarkers. Specifically, it addresses the analysis of amyloidotic neurodegenerative and cancer biomarkers from different proteomics and metabolomics perspectives, depending on the analytical and the sample preparation strategies employed: (i) targeted or untargeted strategies and (ii) top-down or bottom-up strategies (only in the case of proteomics analysis). Furthermore, different microscale separation techniques, such as capillary electrophoresis (CE), capillary liquid chromatography (CapLC) and ion mobility spectrometry (IMS), all of them hyphenated to mass spectrometry (CE-MS, CapLC-MS and IM-MS, respectively) are evaluated for the analysis of different proteins and metabolites of interest.

Transthyretin (TTR) is a homotetrameric protein known to misfold and aggregate causing different types of amyloidosis. Familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis, is associated with a TTR variant that presents a single amino acid substitution of valine for methionine at position 30 of the monomeric sequence (Met30). In this thesis, we describe different off-line and on-line immunoprecipitation (IP) procedures under denaturing and non-denaturing conditions for the isolation of TTR from healthy controls and FAP-I human serum samples prior analysis by CE- MS, CapLC-MS and IM-MS. The obtained results permit us to propose complementary and reliable methods to screen, diagnose, and follow-up patients with suspected TTR amyloidosis, as well as to gain a novel insight into the structural changes on TTR proteoforms or oligomers to understand the mechanism underlying FAP-I.

Huntington’s disease (HD) is also subject of study of this thesis. HD is an inherited neurodegenerative disorder caused by an expansion of the cytosine-adenine-guanine (CAG) repeat in the exon 1 of the huntingtin gene (HTT), which encodes a stretch of glutamines in the huntingtin protein that make it prone to form amyloid fibrils. However, the mechanisms involving aggregation onset or progression are not fully understand yet. Therefore, studies targeting metabolism as an alternative to the huntingtin protein are regarded as necessary. The present thesis describes the optimization of a plasma pretreatment for optimized recovery of low molecular mass compounds prior to on-line solid-phase extraction capillary electrophoresis mass spectrometry with a C18 chromatographic sorbent (C18-SPE-CE-MS), a novel sensitive method for on-line sample clean-up and concentration enhancement in metabolomics studies. Moreover, it presents the capacity of the C18-SPE-CE-MS methodology to analyze low molecular mass compounds in plasma samples from wild-type (wt) and HD mice in combination with advanced chemometric data analysis tools, such as multivariate curve resolution alternating least squares (MCR-ALS), principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), for the identification of potential metabolite biomarker candidates involved in HD progression, which could provide a window of opportunity for prediction of disease onset, evaluation of HD early progression and response to treatment.

Colorectal cancer (CRC) and liver metastatic disease are also studied in this thesis. Specifically, human carcinoembryonic antigen (CEA) is a highly N-glycosylated protein (60% m/m of glycans and 28 N-linked glycosylation sites) found in normal human colonic epithelial cells, as well as in tumor forming CRC cell lines. Despite the demonstrated usefulness of CEA as a biomarker for monitoring CRC and liver metastatic disease patients’ therapy, improvement in the selectivity of CEA detection remains a challenge for clinical tumor diagnosis. This thesis describes a multienzyme sheathless capillary electrophoresis tandem mass spectrometry (CE- MS/MS) strategy for the N-glycosylation analysis of CEA samples purified from human colon carcinoma and human liver metastases. The obtained results permit us to propose novel potential N-glycopeptide markers, which could provide important biologic information on how N-glycosylation may influence CEA structure in CRC and liver metastatic disease.