Nuevas estrategias de espectrometría de masas para la obtención y cuantificación de bioimágenes de platino y determinación de proteínas ligadas a fármacos platinados en estudios de nefrotoxicidad

Desde que en los años 70 se descubriese fortuitamente la actividad antineoplásica del cisplatino, éste ha sido empleado con gran éxito en el tratamiento de numerosos tumores, siendo hoy en día el fármaco más empleado en quimioterapia. Sin embargo, en su aplicación surgen diversos inconvenientes como la aparición de resistencias intrínsecas o desarrolladas al fármaco, así como la aparición de diferentes efectos secundarios. Entre estos últimos destaca la nefrotoxicidad inducida en el tratamiento con cisplatino, la cual limita la dosis administrable del fármaco. Por ello, otros fármacos con base de Pt de segunda y tercera generación han sido desarrollados con el fin de mejorar sus propiedades. Sin embargo, actualmente el cisplatino sigue siendo ineludible en las terapias contra el cáncer. Es bien sabido que la actividad antineoplásica del cisplatino se debe a su interacción con su diana farmacológica principal, el ADN, al cual se coordina formando diferentes aductos cisplatino-ADN que provocan una disrupción en la doble hélice y provocando así la muerte celular por apoptosis. Sin embargo, debido a la gran reactividad que presenta el cisplatino, éste puede unirse a otras biomoléculas diferentes a su diana farmacológica como son las proteínas. Esta interacción es de gran importancia, ya que juega un papel fundamental en su complicado mecanismo de toxicidad y en la aparición de resistencia al fármaco y efectos secundarios. Por ello, el estudio de dichas interacciones presenta gran interés, ya que puede arrojar cierta luz a la comprensión de los mecanismos de aparición de nefrotoxicidad, lo cual ayudaría en el diseño de terapias mejoradas y fármacos nefroprotectores. Por todo ello, el objetivo principal de esta tesis doctoral ha consistido en el desarrollo de estrategias bioanalíticas basadas en espectrometría de masas atómica y molecular, técnicas de separación electroforéticas y nuevos métodos de preparación de la muestra para la determinación de proteínas ligadas a fármacos con base de Pt en muestras biológicas relacionadas con el fracaso renal, así como para la obtención de mapas de distribución de Pt en tejidos afectados..

Etiologies and outcomes of acute liver failure in a spanish community

Previous retrospective study (1992 to 2000) performed in Spain showed that drug toxicity, viral hepatitis, and indeterminate etiology were the most prevalent causes of acute liver failure (ALF). In the last decade, there is no information about ALF in our country. For these reasons we analyze retrospectively, in a ten-year period (2000 to 2010), the presumed causes, clinical characteristics, course, and outcome of ALF in a Spanish community. Causes of ALF were indeterminate in 4 patients (24%), acute hepatitis B infection in 4 patients (24%), drug or toxic reactions in 4 patients (24%), including one case of acetaminophen overdose, followed by miscellaneous causes. The overall short-term survival (6 weeks after admission) was 65%. Liver transplantation was performed in 11 patients with a survival of 82%. Despite fulfilling criteria, 2 patients were not transplanted because of contraindications; they both died. In summary, acute hepatitis B and indeterminate cause are still being the most frequent causes of ALF in our region, and patients with ALF have an excellent chance of survival after emergency liver transplantation. Acetaminophen overdose still represents a very rare cause of ALF in our community

Curso abierto de ayuda para la elaboración del Trabajo Fin de Grado en los Grados en Química e Ingeniería Química

Este proyecto llevará a cabo el desarrollo de un curso abierto que sirva a los estudiantes como guía de ayuda a la hora de elaborar su Trabajo Fin de Grado (TFG) en los Grados de Química e Ingeniería Química. Se incluirá una descripción de herramientas que los estudiantes puedan emplear en el desarrollo de sus TFG. Éstas serán seleccionadas de acuerdo con la experiencia de los miembros del Grupo y en función de las necesidades de los estudiantes. El curso resultante se editará para libre acceso a través del portal iTunes U, de ámbito internacional, convirtiéndose en el primer curso UCM ofertado en esta plataforma (tras consulta y aprobación del Vicerrectorado de Innovación; de no ser así, se alojará en alguna otra plataforma de amplia difusión). Se aprovechará la accesibilidad ofertada a los estudiantes por iTunes U para poner a su disposición el material elaborado logrando una gran difusión, incrementándose la visibilidad internacional de la UCM. El material de ayuda elaborado para el curso se presentará tanto en español como en inglés, para facilitar su uso por alumnos visitantes de la UCM, favoreciendo la movilidad de los estudiantes en el marco del EEES. Este material también se adaptará para poder ser empleado de forma directa, totalmente o en forma de módulos, en el Campus Virtual UCM

Bridging the gap between molecular and elemental mass spectrometry: Higher energy collisional dissociation (HCD) revealing elemental information

Molecular mass spectrometry has been applied to simultaneously obtain molecular and elemental information from metal-containing species. Energy tuning of the higher-energy collision dissociation (HCD) fragmentation cell allows the controlled production of typical peptide fragments or elemental reporter ions informing about the metallic content of the analyzed species. Different instrumental configurations and fragmentation techniques have been tested, and the efficiency extracting the elemental information has been compared. HCD fragmentation operating at very high energy led to the best results. Platinum, lanthanides, and iodine reporter ions from peptides interacting with cisplatin, peptides labeled with lanthanides-MeCAT-IA, and iodinated peptides, respectively, were obtained. The possibility to produce abundant molecular and elemental ions in the same analysis simplifies the correlation between both signals and open pathways in metallomics studies enabling the specific tracking of metal-containing species. The proposed approach has been successfully applied to in solution standards and complex samples. Moreover, interesting preliminary MALDI-imaging experiments have been performed showing similar metal distribution compared to laser ablation (LA)-ICPMS

OFFGEL isoelectric focusing and polyacrylamide gel electrophoresis separation of platinum-binding proteins

In this work a 2D electrophoretic separation procedure able to maintain the integrity of platinum–protein bonds has been developed. The method is based on the use of sequential OFFGEL isoelectric focussing (IEF) and PAGE. A systematic study of the reagents used for PAGE, for OFFGEL-IEF separation, and postseparation treatment of gels (such as enzymatic digestion and sample preparation for MS analysis) was tackled regarding their suitability for the identification of platinum binding proteins using standard proteins incubated with cisplatin. The distribution of platinum in high and low molecular weight fractions (separated by cut-off filters) was determined by ICP-MS, which allows evaluating platinum–protein bond stability under the conditions studied. SDS-PAGE in the absence of -mercaptoethanol or dithiotreitol preserved the platinum–protein bonds. In addition, neither the influence of the electric field during the electrophoretic separation, nor the processes of fixing, staining and destaining of proteins in the gel did result in the loss of platinum from platinum binding proteins. SDS-PAGE under non-reducing conditions provides separation of platinum-binding proteins in very narrow bands with quantitative recoveries. Different amounts of platinum-bound proteins covering the range 0.3–2.0 g were separated and mineralised for platinum determination, showing good platinum linearity. Limits of detection for a mixture of five standard proteins incubated with cisplatin were between the range of 2.4 and 13.9 pg of platinum, which were satisfactory for their application to biological samples. Regarding OFFGEL-IEF, a denaturing solution without thiourea and without dithiotreitol is recommended. The suitability of the OFFGEL-IEF for the separation of platinum binding proteins of a kidney cytosol was demonstrated

A shotgun approach for the identification of platinum–protein complexes

A shotgun approach including peptide-based OFFGEL-isoelectric focusing (IEF) fractionation has been developed with the aim of improving the identification of platinum-binding proteins in biological samples. The method is based on a filter-aided sample preparation (FASP) tryptic digestion under denaturing and reducing conditions of cisplatin–, oxaliplatin–, and carboplatin–protein complexes, followed by OFFGEL-IEF separation of the peptides. Any risk of platinum loss is minimized throughout the procedure due to the removal of the reagents used after each stage of the FASP method and the absence of thiol-based reagents in the focusing buffer employed in the IEF separation. The platinum–peptide complexes stability after the FASP digestion and the IEF separation was confirmed by size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS). The suitability of peptide-based OFFGEL-IEF fractionation for reducing the sample complexity for further nano-liquid chromatographyelectrospray ionization-tandem mass spectrometry (nLC-ESIMS/MS) analysis has been demonstrated, allowing the detection of platinum-containing peptides, with significantly lower abundance and ionization efficiency than unmodified peptides. nLCMS/MS analysis of selected OFFGEL-IEF fractions from tryptic digests with different complexity degrees: standard human serum albumin (HSA), a mixture of five proteins (albumin, transferrin, carbonic anhydrase, myoglobin, and cytochrome-c) and human blood serum allowed the identification of several platinum–peptides from cisplatin–HSA. Cisplatin-binding sites in HSA were elucidated from the MS/MS spectra and assessed considering the protein three-dimensional structure. Most of the potential superficial binding sites available on HSA were identified for all the samples, including a biologically relevant cisplatin-cross-link of two protein domains, demonstrating the capabilities of the methodolog

Bridging the Gap between Molecular and Elemental Mass Spectrometry: Higher Energy Collisional Dissociation (HCD) Revealing Elemental Information

Molecular mass spectrometry has been applied to simultaneously obtain molecular and elemental information from metal-containing species. Energy tuning of the higher-energy collision dissociation (HCD) fragmentation cell allows the controlled production of typical peptide fragments or elemental reporter ions informing about the metallic content of the analyzed species. Different instrumental configurations and fragmentation techniques have been tested, and the efficiency extracting the elemental information has been compared. HCD fragmentation operating at very high energy led to the best results. Platinum, lanthanides, and iodine reporter ions from peptides interacting with cisplatin, peptides labeled with lanthanides-MeCAT-IA, and iodinated peptides, respectively, were obtained. The possibility to produce abundant molecular and elemental ions in the same analysis simplifies the correlation between both signals and open pathways in metallomics studies enabling the specific tracking of metal-containing species. The proposed approach has been successfully applied to <i>in solution</i> standards and complex samples. Moreover, interesting preliminary MALDI-imaging experiments have been performed showing similar metal distribution compared to laser ablation (LA)-ICPMS