Desarrollo de nuevos sistemas separativos para la determinación de biomoléculas en matrices vegetales

La presente memoria se enmarca dentro de las líneas de investigación del grupo de investigación, entre las que se encuentran: i) el desarrollo de materiales poliméricos con fines separativos, y ii) la puesta a punto de metodologías analíticas rápidas y fiables para el control de calidad en la industria de aceites vegetales y otros productos de interés agroalimentario. En consecuencia, los objetivos principales de esta Tesis Doctoral son: i) el diseño y optimización de soportes poliméricos modificados con nanopartículas metálicas (tales como el oro y la plata) para la extracción de proteínas en matrices vegetales, ii) la preparación y evaluación de sistemas de extracción de proteínas en dichas muestras asistida por enzimas y iii) el desarrollo de metodologías analíticas de separación rápidas y fiables con el fin de poder establecer el origen genético y botánico de diversos productos alimentarios. La presente memoria de Tesis Doctoral se divide en cuatro grandes bloques. El primer bloque consta de una introducción, donde se describen brevemente las muestras vegetales empleadas en esta Tesis Doctoral (fruto, hoja y aceite de olivo, frutos de la variedad Citrus (en concreto naranjas y mandarinas) y muérdago, correspondientes a los Capítulos 1-3, respectivamente). Además, se incluye una revisión actualizada de los métodos de extracción empleados durante la tesis: extracción en fase sólida (SPE), extracción asistida por enzimas y por último, el empleo de las bibliotecas combinatorias de ligandos peptídicos (CPLLs). El bloque de introducción concluye con una breve descripción de las técnicas analíticas empleadas en el desarrollo de la Tesis Doctoral (Capítulo 5). El segundo bloque (que engloba los capítulos 6-11) está dedicado al desarrollo de nuevas técnicas de extracción de proteínas en matrices de origen vegetal. Para ello, se han puesto a punto nuevos sorbentes, basados en materiales poliméricos sintetizados a partir del metacrilato de glicidilo (GMA), que son posteriormente modificados con nanopartículas metálicas (de oro y plata) para su uso como sorbentes en SPE. Por otro lado, la presencia de sistemas reticulares u organizados, presentes en los vegetales, requiere habitualmente de tratamientos relativamente “agresivos” con el fin de poder extraer los analitos diana. En este sentido, se han llevado a cabo estudios de extracción de proteínas asistidos por enzimas, empleándose tanto enzimas individuales como mezclas de las mismas. Gracias a la acción de estas biomacromoléculas, se facilita la ruptura de las membranas celulares, y por ende la extracción de los analitos para su posterior análisis y cuantificación. Estos pretratamientos de la muestra, sin duda, permitirán obtener resultados de mayor calidad. En este caso, se ha empleado dicha tecnología para la extracción de proteínas de diversos productos del olivo (hojas, huesos y pulpa), así como para muestras de naranja y mandarina, incluyendo tanto la piel como la pulpa de las mismas. Por último, se ha llevado a cabo un estudio del proteoma del muérdago mediante la extracción del mismo empleando las CPLLs, que son capaces de extraer y preconcentrar las proteínas de baja abundancia. La identificación del mapa proteico de esta planta ha permitido profundizar en un mayor conocimiento de sus propiedades medicinales. El tercer bloque (que engloba los capítulos 12-20) muestra el desarrollo de una amplia variedad de métodos de análisis para la determinación de diferentes compuestos en matrices de origen vegetal, tales como aceite de oliva, frutos y hojas de olivo, frutos de la variedad Citrus (naranjas y mandarinas), y muérdago). Dichas metodologías abarcan tanto técnicas de carácter cromatográfico (HPLC) y afines (CZE, CGE y CEC) así como de carácter espectrométrico (ATR-FTIR o DIMS). Se han desarrollado metodologías analíticas para la determinación de proteínas, TAGs, esteroles, ácidos grasos y péptidos. Además, en muchos de los capítulos, se han aplicado herramientas quimiométricas de análisis, en concreto, se han construido modelos de análisis discriminante lineal (LDA), para establecer la clasificación de muestras de aceites vegetales en función de su origen botánica, para la discriminación de productos del olivo, incluyendo aceites de oliva, en función de su variedad genética y en caso de los aceites de oliva, también para clasificarlos en función de su índice de madurez. En algunos de ellos, se ha aplicado la regresión lineal múltiple (MLR) para cuantificar mezclas binarias de diferentes variedades genéticasThis PhD thesis reports the development of novel separation systems to determine biomolecules from complex matrices, especially in vegetal samples. In the first part of this thesis, extraction methodologies were designed to isolate proteins, constituting a significant contribution in the sample preparation field. Thus, the development of novel in-house sorbents based on organic polymers modified with Au and AgNPs for SPE with improved properties, the proper selection of enzyme preparations (either alone or in mixtures) for efficient release of these biomacromolecules from plant cells, and the use of CPLLs to preconcentrate LAPs in complex matrices have been accomplished. Significant enhancements such as large protein extraction yield, tunable selectivity, cost-effective, environmentally friendly and other advantages should place the developed methodologies in a competitive position compared to commercial SPE materials or other traditional extraction methodologies (that imply the use of organic solvents). Another part covered by this thesis was the development of methods for the characterization and determination of several compounds in olive products using different chromatographic and spectrometric techniques. For this purpose, different profiles belong to components present in these matrices, such as proteins, TAGs, sterols and fatty acids were considered. These profiles allowed a discrimination of vegetable oils according to their botanical origin, and also the characterization and authentication of Spanish and Tunisian olive oils in relation to their genetic variety. The proposed methodologies are of utmost interest to assure product quality and to investigate fraudulent practices in food science. In this section of the PhD Thesis, and as required by the aforementioned regulation of the University of Valencia, a summary of the results shown in the successive articles jointly with Sections II and III and the most relevant conclusions of each work is briefly presented here

Accurate determination of the milk protein allergen β-lactoglobulin by on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry

An on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS) method was developed to purify, preconcentrate, separate, and characterize the milk allergenic protein β-lactoglobulin (β-LG) in food samples. The sorbent to pack into the SPE microcartidges was prepared by immobilizing an aptamer against β-LG onto magnetic bead particles. After optimizing the SPE-CE-MS method, the sample (ca. 75 µL) was loaded in separation background electrolyte (BGE, 2 M acetic acid pH 2.2), while a solution of 100 mM NH4OH (pH 11.2) (ca. 100 nL) was used for the protein elution. The linearity of the method ranged between 0.1 and 20 µg·mL-1 and the limit of detection (LOD) was 0.05 µg·mL-1, which was 200 times lower than by CE-MS. The method was repeatable in terms of relative standard deviation (RSD) for migration times and peak areas (< 0.5 % and 2.4 %, respectively) and microcartridge lifetime was more than 25 analyses. The applicability of the method for the determination of low levels of β-LG was shown by analyzing milk-free foods (i.e. a 100 % cocoa dark chocolate, a hypoallergenic formula for infants, and a dairy-free white bread) and milk-containing white breads. Results were satisfactory in all cases, thus demonstrating the great potential of the developed method for accurate food safety and quality control

Determination of a lectin protein allergen in food by on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry

An aptamer affinity sorbent was prepared for clean-up, preconcentration, separation and characterization of a food allergen protein by on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS). SPE microcartridges were packed with a sorbent based on magnetic bead particles modified with an aptamer against the target lectin protein concanavalin A (Con A). After optimization of several parameters of the SPE-CE-MS method, the sample (ca. 30 µL) was loaded in separation background electrolyte (BGE, 2 M acetic acid pH 2.2), while the retained protein was eluted with 100 mM NH4OH (pH 11.2) (ca. 100 nL). The developed method was linear between 0.5 and 20 mg·L-1 and the limit of detection (LOD) was 0.25 mg·L-1, which was 100 times lower than by CE-MS. The repeatability of the method was satisfactory, with relative standard deviations (RSD) for migration times and peak areas below 1.9 and 8.1%, respectively. In addition, the microcartridges could be reused more than 25 analyses without significant loss of extraction efficiency. Finally, the applicability and versatility of the developed method were demonstrated by analyzing low levels of Con A in different food matrices (i.e. white beans, as well as chickpea, lentils, and wheat flours), leading to satisfactory results, with recoveries between 87 and 115%

Preclinical and randomized phase I studies of plitidepsin in adults hospitalized with COVID-19

Plitidepsin, a marine-derived cyclic-peptide, inhibits SARS-CoV-2 replication at nanomolar concentrations by targeting the host protein eukaryotic translation elongation factor 1A. Here, we show that plitidepsin distributes preferentially to lung over plasma, with similar potency against across several SARS-CoV-2 variants in preclinical studies. Simultaneously, in this randomized, parallel, open-label, proof-of-concept study (NCT04382066) conducted in 10 Spanish hospitals between May and November 2020, 46 adult hospitalized patients with confirmed SARS-CoV-2 infection received either 1.5 mg (n = 15), 2.0 mg (n = 16), or 2.5 mg (n = 15) plitidepsin once daily for 3 d. The primary objective was safety; viral load kinetics, mortality, need for increased respiratory support, and dose selection were secondary end points. One patient withdrew consent before starting procedures; 45 initiated treatment; one withdrew because of hypersensitivity. Two Grade 3 treatment-related adverse events were observed (hypersensitivity and diarrhea). Treatment-related adverse events affecting more than 5% of patients were nausea (42.2%), vomiting (15.6%), and diarrhea (6.7%). Mean viral load reductions from baseline were 1.35, 2.35, 3.25, and 3.85 log10 at days 4, 7, 15, and 31. Nonmechanical invasive ventilation was required in 8 of 44 evaluable patients (16.0%); six patients required intensive care support (13.6%), and three patients (6.7%) died (COVID-19-related). Plitidepsin has a favorable safety profile in patients with COVID-19.This work was supported by grants from the Government of Spain (PIE_INTRAMURAL_ LINEA 1 - 202020E079; PIE_INTRAMURAL_CSIC-202020E043). The research of CBIG consortium (constituted by IRTA-CReSA, BSC, & IrsiCaixa) is supported by Grifols pharmaceutical. We also acknowledge the crowdfunding initiative #Yomecorono (https://www.yomecorono.com). N Izquierdo-Useros has nonrestrictive funding from PharmaMar to study the antiviral effect of Plitidepsin. NJ Krogan was funded by grants from the National Institutes of Health (P50AI150476, U19AI135990, U19AI135972, R01AI143292, R01AI120694, and P01AI063302); by the Excellence in Research Award (ERA) from the Laboratory for Genomics Research (LGR), a collaboration between the University of California, San Francisco (UCSF), University of California, Berkley (UCB), and GlaxoSmithKline (GSK) (#133122P); by the Roddenberry Foundation, and gifts from QCRG philanthropic donors. This work was supported by the Defense Advanced Research Projects Agency (DARPA) under Cooperative Agreement #HR0011-19-2-0020. The views, opinions, and/or findings contained in this material are those of the authors and should not be interpreted as representing the official views or policies of the Department of Defense or the U.S. Government. This research was partly funded by Center for Research for Influenza Pathogenesis and Transmission (CRIPT), a National Institute of Allergy and Infectious Diseases (NIAID) supported Center of Excellence for Influenza Research and Response (CEIRS, contract # 75N93021C00014), by DARPA grant HR0011-19-2-0020, by supplements to NIAID grants U19AI142733, U19AI135972, and DoD grant W81XWH-20-1-0270, and by the generous support of the JPB Foundation, the Open Philanthropy Project (research grant 2020-215611 (5384)), and anonymous donors to A García-Sastre. S Yildiz received funding from a Swiss National Foundation Early Postdoc Mobility fellowship (P2GEP3_184202).Peer reviewe

On-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry for the analysis of protein biomarkers in biological fluids and food: A tutorial

The analysis by capillary electrophoresis (CE) of low abundant proteins in complex samples, such as biological fluids and food, is especially challenging, due to the poor concentration sensitivity of microscale separation tech- niques and the sample matrix complexity. In order to overcome these major drawbacks, microextraction sample preparation techniques based on on-line solid-phase extraction capillary electrophoresis (SPE-CE) are regarded as an excellent alternative for sample matrix clean-up and analyte preconcentration with minimum sample han- dling. In this study, we present, as a tutorial, a valve-free on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS) method for purification, preconcentration, separation, de- tection, and characterization of intact protein biomarkers in biological fluids and food using as cases of study ‑synuclein ( ‑syn), concanavalin A (Con A), and -lactoglobulin ( -LG), which are related to Parkinson's dis- ease and food allergy, respectively. This tutorial is not limited to the description of the analytical method, but it also provides ready-to-use preparation procedures for sorbent and microextraction devices, and introduces strategies to overcome undesired effects, allowing a straightforward implementation and optimum performance of AA-SPE-CE-MS, as a platform to develop further application

Aptamer-functionalized magnetic supports for sample preparation

In the last few years, aptamer-functionalized materials have been used as promising affinity sorbents for sample preparation purposes. These selective materials have attracted much attention due to their excellent molecular recognition properties, high stability, and the possibility of incorporation onto the surface of different supporting materials, such as nanoparticles, polymers, etc. On the other hand, current trends in extraction techniques encompass the use of miniaturized devices integrated with advanced affinity-based sorbents. Within these sample preparation devices, magnetic nanoparticles have become excellent candidates for the preparation of new functional nanomaterials for the solid-phase extraction processes. In addition to this approach, magnetic elements can be integrated in extraction devices (e.g., stir bars, stir cakes, etc.) to provide efficient extraction/stirring integrated techniques able to simplify the extraction and improve the performance. This review gives an overview of the literature published regarding aptamer-based sorbents using as host supports magnetic nanoparticles and integrated stirred units. For this purpose, the most relevant developments and applications achieved in the last seven years (from 2017 to 2023) in sample preparation by the integration of these affinity-based materials in extraction techniques such as magnetic solid-phase extraction and stir bar sorptive extraction will be reviewed

Galactose-functionalized methacrylate polymers as affinity sorbents for extraction of food allergen lectins

In this study, glycidyl methacrylate (GMA)-based materials functionalized with different galactose derivatives were prepared to be used as affinity sorbents for solid-phase extraction (SPE) of several food allergen lectins (such as phytohemagglutinin (PHA)). First, GMA-based polymers were synthesized and then galactose derivatives were immobilized onto the GMA surface using two different synthetic routes. In the first approach, the bare polymer was modified with ethylenediamine and glutaraldehyde, and subsequently two galactose derivatives were immobilized. In the second strategy, the starting polymer was modified with cystamine and gold nanoparticles (AuNPs), on which a thiolated galactose derivative was subsequently anchored. The resulting materials were characterized by scanning electron microscopy and used as SPE sorbents for the isolation of PHA (as probe protein) from food matrices. Different SPE parameters (sample pH, eluent solution composition, binding capacity, sample volume, selectivity and reusability) were evaluated. The material that provided the best PHA recovery (98%) was the one obtained in the second approach, being this material successfully applied to the selective extraction of PHA and other similar lectins from different foods (red and lima dried beans, fresh soybeans and biscuits containing soybean protein traces as indicated in their label). After SDS-PAGE of eluates, all samples only exhibited the characteristic PHA band around 30 kDa, suggesting the high potential of the developed material for application in food allergy field

Use of an enzyme-assisted method to improve protein extraction from olive leaves

The improvement of protein extraction from olive leaves using an enzyme-assisted protocol has been investigated. Using a cellulase enzyme (Celluclast® 1.5L), different parameters that affect the extraction process, such as the influence and amount of organic solvent, enzyme amount, pH and extraction temperature and time, were optimized. The influence of these factors was examined using the standard Bradford assay and the extracted proteins were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum extraction parameters were: 30% acetonitrile, 5% (v/v) Celluclast® 1.5L at pH 5.0 and 55 °C for 15 min. Under these conditions, several protein extracts from olive leaves of different genetic variety (with a total protein amount comprised between 1.87 and 6.64 mg g-1) were analyzed and compared by SDS-PAGE, showing differences in their electrophoretic protein profiles. The developed enzyme-assisted extraction method has shown a faster extraction, higher recovery and reduced solvent usage with respect to the use of the non-enzymatic methods described in literature

Use of protein profiles established by CZE to predict the cultivar of olive leaves and pulps

Intact protein profiles established by CZE have been used to predict the cultivar of olive leaves and pulps. For this purpose, proteins were extracted using a mild enzyme-assisted extraction method, which provided higher protein recoveries and a lower environmental impact than other previously described methods. These extracts were subjected to CZE determination under basic conditions using a BGE composed of 50 mM phosphate, 50 mM tetraborate and 0.1% PVA at pH 9. Nine and 14 common peaks, for leaf and pulp samples, respectively, were identified in the nine cultivars studied in this work. In addition, and using linear discriminant analysis of the CZE data, olive leaf and pulp samples belonging to nine cultivars from different Spanish regions were correctly classified with an excellent resolution among all categories, which demonstrated that intact protein profiles are characteristic of each cultiva

Polymeric sorbents modified with gold and silver nanoparticles for solid-phase extraction of proteins followed by MALDI-TOF analysis

The authors describe four different kinds of sorbents for solid-phase extraction (SPE) and preconcentration of proteins from complex samples. All are based on the use of a poly(glycidyl-co-ethylene dimethacrylate) host monolith that was chemically functionalized by using two different ligands (ammonia and cysteamine). Gold nanoparticles (AuNPs) or silver NPs were then assembled to the amino or thiol groups. The resulting materials are shown to be viable stationary phases for use in SPE cartridges. The sorbents can selectively retain bovine serum albumin, and the thiol-modified sorbents containing AuNPs and AgNPs provide the highest recoveries (>90%) and satisfactory loading capacities (29.3 and 17.6 μg⋅mg−1 of sorbent, respectively). The applicability of these nanosorbents was demonstrated by preconcentrating viscotoxins from mistletoe extracts. The enriched fractions were subjected to MALDI-TOF analysis to underpin their selectivity