Efficient Extraction of Olive Pulp and Stone Proteins by using an Enzyme-Assisted Method
An efficient protein extraction protocol for proteins from olive pulp and stone by using enzymes was developed. For this purpose, different parameters that affect the extraction process, such as enzyme type and content, pH, and extraction temperature and time, were tested. The influence of these factors on protein recovery was examined using the standard Bradford assay, while the extracted proteins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The best extraction conditions were achieved at pH 7.0 and 5% (v/v) Palatase® 20000 L (lipase) for pulp and Lecitase® Ultra (phospholipase) for stone proteins. The optimal extraction temperature and time w…
Capillary Electrophoresis of Free Fatty Acids by Indirect Ultraviolet Detection: Application to the Classification of Vegetable Oils According to Their Botanical Origin
A method for the determination of fatty acids in vegetable oils by capillary electrophoresis with indirect UV–vis detection has been developed. The separation of fatty acids was optimized in terms of Brij surfactant nature and concentration and organic modifier (2-propanol) percentage. The optimal background electrolyte consisted of 10 mM p-hydroxybenzoate, 5 mM Tris at pH 8.8, 80 mM Brij 98, 40% acetonitrile, and 10% 2-propanol. Under these conditions, vegetable oils from five botanical origins (avocado, corn, extra virgin olive, hazelnut, and soybean) were analyzed and the fatty acid contents established. Linear discriminant analysis (LDA) models were constructed using fatty acid peak are…
Smart Materials for Solid-Phase Extraction Applications
New In-Depth Analytical Approach of the Porcine Seminal Plasma Proteome Reveals Potential Fertility Biomarkers
A complete characterization of the proteome of seminal plasma (SP) is an essential step to understand how SP influences sperm function and fertility after artificial insemination (AI). The purpose of this study was to identify which among characterized proteins in boar SP were differently expressed among AI boars with significantly different fertility outcomes. A total of 872 SP proteins, 390 of them belonging specifically to Sus Scrofa taxonomy, were identified (Experiment 1) by using a novel proteomic approach that combined size exclusion chromatography and solid-phase extraction as prefractionation steps prior to Nano LC-ESI-MS/MS analysis. The SP proteomes of 26 boars showing significan…
Solid-phase extraction based on ground methacrylate monolith modified with gold nanoparticles for isolation of proteins
In this study, a novel polymeric material functionalized with gold nanoparticles (AuNPs) was prepared as solid-phase extraction (SPE) sorbent for isolation of proteins. The sorbent was synthesized from a powdered poly(glycidyl-co-ethylene dimethacrylate) monolith, and modified with ammonia, followed by immobilization of AuNPs on the pore surface of the material. To evaluate the performance of this SPE support, proteins were selected as test solutes, being the extraction conditions and other parameters (loading capacity and regenerative ability of sorbent) established. The results indicated that this sorbent could be employed to selectively capture proteins according to their pI, on the basi…
In syringe hybrid monoliths modified with gold nanoparticles for selective extraction of glutathione in biological fluids prior to its determination by HPLC.
Abstract In this work, a simple device for extraction glutathione (GSH) in biological fluids using a hybrid monolithic material within a polypropylene syringe is developed. For this purpose, glycidyl methacrylate-based monolith was firstly prepared within this housing material, and the polymer was modified with different ligands (ammonia, cysteamine and cystamine). The resulting materials (containing amine or thiol groups, respectively) were then functionalized with gold nanoparticles (AuNPs). The hybrid material that gave the largest AuNPs coverage was selected as solid-phase (SPE) sorbent and several variables affecting the extraction recovery of this compound were investigated. Under opt…
Classification of olive leaves and pulp extracts by comprehensive two-dimensional liquid chromatography of polyphenolic fingerprints.
Abstract The development of a new comprehensive two-dimensional liquid chromatographic method is described, to obtain the profiles of polyphenolic compounds present in olive (Olea europaea L.) leaves and pulps from different genetic origin. Optimisation of the stationary phase nature, particle size, column length and internal diameter, as well as other separation conditions, was performed. Along the study, three stationary phases (C18, PFP and phenyl) in the first dimension (1D), and five (C18, amide, cyano, phenyl and PFP) in the second dimension (2D) were combined to obtain the maximal number of resolved peaks. The optimised method successfully characterised the presence of 26 and 29 comm…
Polymeric monolithic microcartridges with gold nanoparticles for the analysis of protein biomarkers by on-line solid-phase extraction capillary electrophoresis-mass spectrometry
In this study, polymeric monoliths with gold nanoparticles (AuNP@monolith) were investigated as microcartridges for the analysis of protein biomarkers by on-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS). “Plug-and-play” microcartridges (7 mm) were prepared from a glycidyl methacrylate (GMA)-based monolithic capillary column (5 cm x 250 µm i.d.), which was modified with ammonia and subsequently functionalized with gold nanoparticles (AuNPs). The performance of these novel microcartridges was evaluated with human transthyretin (TTR), which is a protein related to different types of familial amyloidotic polyneuropathies (FAP). Protein retention depended on…
Proteomic fingerprinting of mistletoe (Viscum album L.) via combinatorial peptide ligand libraries and mass spectrometry analysis
Abstract Combinatorial peptide ligand libraries (CPLLs), coupled to mass spectrometry (MS) analysis, have been used to investigate in depth the proteome of Viscum album L. (VA), commonly named European mistletoe, in order to provide a first proteomic fingerprinting. For this purpose, the proteins were captured via CPLLs at two different pH values (acidic and neutral). A total of 648 non-redundant proteins were identified by using two different databases. The two pH values, chosen for bead incubations, have contributed to increment the capture ability: 56% and 31% of CPLLs species were respectively recognized at pH 7.2 and at pH 2.2. Finally the biological function of identified proteins was…
Enzyme-assisted extraction of proteins from Citrus fruits and prediction of their cultivar using protein profiles obtained by capillary gel electrophoresis
Abstract The suitability of protein profiles established by capillary gel electrophoresis (CGE) as a tool to discriminate between 11 cultivars of Citrus (orange and tangerine) peel and pulp was evaluated in this work. Before CGE analysis, different extraction buffers (which included enzyme-assisted treatments) were compared. The best results were achieved using 5% ( v/v ) Celluclast ® 1.5 L and 5% ( v/v ) Palatase ® 20,000 L buffers for Citrus peel and pulp protein extracts, respectively. The resulting protein profiles obtained were used to construct linear discriminant analysis (LDA) models able to distinguish Citrus peel and pulp samples according to their cultivar. In both cases, all sam…
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 belong…
Boronate affinity sorbents based on thiol-functionalized polysiloxane-polymethacrylate composite materials in syringe format for selective extraction of glycopeptides
Abstract In this work, two novel boronate affinity monolithic materials able to extract glycopeptides within a polypropylene syringe are described and compared. The first material was synthesized from glycidyl methacrylate (GMA)-based monoliths modified with poly-3-mercaptopropyl-methylsiloxane (PMPMS) followed by attachment of 4-vinylphenylboronic acid (VPBA) via thiol-ene click reaction. The second material was prepared by using gold nanoparticle (AuNP)-modified monoliths as substrate followed by subsequent attachment of PMPMS and VPBA. The resulting materials were used as sorbents for solid-phase extraction (SPE) to selectively preconcentrate glycopeptides from horseradish peroxidase (HR…
Recent advances in aptamer-based miniaturized extraction approaches in food analysis
Abstract Sample preparation is a relevant step in food analysis to achieve adequate extraction and preconcentration of target analytes before their introduction in the analytical system. Current trends in sample preparation involve moving towards miniaturized extraction devices combined with advanced affinity-based sorbents. Within affinity-based materials, aptamer-based ones have attracted much attention due to their excellent molecular recognition properties, high stability, and the possibility of immobilization onto the surface of different supporting materials, such as nanoparticles, monolithic stationary phases, etc. This review gives an overview of the literature published regarding a…
Poly(ethylene glycol) diacrylate based monolithic capillary columns for the analysis of polar small solutes by capillary electrochromatography
Monolithic stationary phases based on poly(ethylene glycol) diacrylates for capillary electrochromatography were developed. Several poly(ethylene glycol) diacrylates (Mn 250, 575, and 700) were used as single monomers and the resulting columns were carefully compared. Methanol and ethyl ether were selected as porogenic solvents, and in all cases ultraviolet radiation was selected as initiation method to prepare polymeric monoliths. The influence of the monomer chain length and ratio monomer/porogen on the morphological and electrochromatographic properties of the resulting monoliths was investigated. Several families of compounds with different polarity (alkyl benzenes, organophosphorous pe…
Determination of antibiotics in meat samples using analytical methodologies: A review
Antibiotics are widely used to prevent or treat some diseases in human and veterinary medicine and also as animal growth promoters. The presence of these compounds in foods derived from food-producing animals can be a risk for human health. Consequently, regulatory agencies have set maximum residue limits for antibiotics in food samples. Therefore, the development of novel methodologies for its determination in food samples is required. Specifically, the analysis and quantification of these substances in meat tissues is a challenge for the analytical chemistry research community. This is due to the complexity of the matrix and the low detection limits required by the regulatory agencies. In…
Poly(ethylene glycol) diacrylate-based solid-phase extraction for determination of sulfonamides in meat samples
Abstract In this work, a sorbent based on poly(ethylene glycol) diacrylate (PEGDA) polymer has been synthesized for solid-phase extraction (SPE) of sulfonamides (sulfaguanidine, sulfadiazine, sulfathiazole, sulfapyridine, sulfamethazine, sulfamonomethoxine, sulfamethoxazole, sulfisoxazole) from different meat matrices, which were subsequently determined by HPLC-DAD. Several extraction parameters such as loading and elution solvents as well as other variables (loading capacity, breakthrough volume and reusability) influencing on the analytical performance of the sorbent were optimized. Under the optimal conditions, the developed method was successfully applied to determine the eight sulfonam…
Current trends in affinity-based monoliths in microextraction approaches: A review
Abstract This article reviews the research contributions along the past five years concerning to monolithic materials for the development of affinity-based sorbents in the field of microextraction techniques. The first part of this paper includes an introduction regarding monolithic affinity media and information of different binding agents (such as immunoglobulin-binding proteins, enzymes, lectins, antibodies, aptamers, dyes and immobilized metal ions and nanoparticles, among others) that can act as affinity ligands. Then, the preparation of monoliths and ligand immobilization strategies as well as the different available formats (syringes, pipette tips, spin columns, capillaries, disks an…
Acrylate ester-based monolithic columns for capillary electrochromatography separation of triacylglycerols in vegetable oils
Abstract A simple and reliable method for the evaluation of triacylglycerols (TAGs) in vegetable oils by capillary electrochromatography (CEC) with UV–Vis detection, using octadecyl acrylate (ODA) ester-based monolithic columns, has been developed. The percentages of the porogenic solvents in the polymerization mixture, and the mobile phase composition, were optimized. The optimum monolith was obtained at the following ratios: 40:60% (wt/wt) monomers/porogens, 60:40% (wt/wt) ODA/1,3-butanediol diacrylate and 23:77% (wt/wt) 1,4-butanediol/1-propanol (14 wt% 1,4-butanediol in the polymerization mixture). A satisfactory resolution between TAGs was achieved in less than 12 min with a 65:35 (v/v…
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 optimised. The influence of these factors was examined using the standard Bradford assay and the extracted proteins were characterised by sodium dodecyl sulphate 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 15min. Under these conditions, several protein extracts…