Identification of avocado (Persea americana) pulp proteins by nano‐LC‐MS/MS via combinatorial peptide ligand libraries

Avocado (Persea americana) proteins have been scarcely studied despite their importance, especially in food related allergies. The proteome of avocado pulp was explored in depth by extracting proteins with capture by combinatorial peptide ligand libraries at pH 7.4 and under conditions mimicking reverse‐phase capture at pH 2.2. The total number of unique gene products identified amounts to 1012 proteins, of which 174 are in common with the control, untreated sample, 190 are present only in the control and 648 represent the new species detected via combinatorial peptide ligand libraries of all combined eluates and likely represent low‐abundance proteins. Among the 1012 proteins, it was possible to identify the already known avocado allergen Pers a 1 and different proteins susceptible to be allergens such as a profilin, a polygalacturonase, a thaumatin‐like protein, a glucanase, and an isoflavone reductase like protein.     

Chicken egg yolk cytoplasmic proteome,mined via combinatorial peptide ligand libraries

The use of combinatorial peptide ligand libraries (CPLLs), containing hexapeptides terminating with a primary amine, or modified with a terminal carboxyl group, or with a terminal tertiary amine, allowed discovering and identifying a large number of previously unreported egg yolk proteins. Whereas the most comprehensive list up to date [K. Mann, M. Mann, Proteomics, 8 (2008) 178–191] tabulated about 115 unique gene products in the yolk plasma, our findings have more than doubled this value to 255 unique protein species. From the initial non-treated egg yolk it was possible to find 49 protein species; the difference was generated thanks to the use of the three combined CPLLs. The aberrant behaviour of some proteins, upon treatment via the CPLL method, such as proteins that do not interact with the library, is discussed and evaluated. Simplified elution protocols from the CPLL beads are taken into consideration, of which direct elution in a single step via sodium dodecyl sulphate desorption seems to be quite promising. Alternative methods are suggested. The list of egg yolk components here reported is by far the most comprehensive at present and could serve as a starting point for isolation and functional characterization of proteins possibly having novel pharmaceutical and biomedical applications.     

Determination of sugars and alcohols in apple juice and cider by high performance liquid chromatography

Summary The application of a high-performance liquid chromatographic produre to the separation and determination of major sugars, sorbitol, glycerol and ethanol in apples, apple juice and cider is described. The HPLC system consisted of a cation-exchange resin column in calcium form, a solvent system of an aqueous solution with calcium EDTA and a refractive index detector. The analysis was completed after 16 minutes. A recovery greater than 91% was observed for all compounds with most recoveries nearing 100%. The coefficients of variation ranged from 2% to 6%.     

Screening of inhibitors for influenza A virus using high-performance affinity chromatography and combinatorial peptide libraries

The affinity inhibitor of fusion peptide of influenza A virus has been studied using a combination of high-performance affinity chromatography (HPAC) and combinatorial peptide libraries. Fusion peptide (FP) (1-11) of influenza A virus was used as the affinity ligand and immobilized onto the poly(glycidyl methacrylate) (PGMA) beads. Positional scanning peptide libraries based on antisense peptide strategy and extended peptide libraries were designed and synthesized. The screening was carried out at acidic pH (5.5) in order to imitate the environment of virus fusion. A hendecapeptide FHRKKGRGKHK was identified to have a strong affinity to the FP (1-11). The dissociation constant of the complex of the hendecapeptide and the FP (1-11) is 3.10 x 10(-6) mol l(-1) in a physiological buffer condition. The polypeptide has a fairly inhibitory effect on three different strains of influenza A virus H1N1 subtype.     

Characterization and analysis of amino acids in orange juice by HPLC-MS/MS for authenticity assessment

Twenty protein α-amino acids have been used to detect adulterations in orange juices from Spanish oranges. An analytical method based on the use of normal-phase liquid chromatography coupled to mass spectrometry has been performed for detection and quantification of these compounds in this complex matrix. MS and MS/MS parameters were optimized and most abundant product ion of each amino acid were selected to be monitorized. Method performance is improved with the use of an Allure Acidix column in which mobile phase composition and flux were optimized. Good separation was achieved using water/acetonitrile (20:80) and water/methanol (10:90) at pH 3, with elution gradient. The method has been used to assess the presence of amino acids in different kinds of orange juice: packing orange juice from both frozen concentrates and recently squeezed oranges, and fresh sweet orange juice hand-squeezed in the laboratory just before the analysis. The differences in composition between the juices and the potential adulteration practices have been evaluated and discussed.     

Application of an ultrasound-assisted surfactant-enhanced emulsification microextraction method for the analysis of diethofencarb and pyrimethanil fungicides in water and fruit juice samples

In this work, a simple, practical and environmentally friendly sample pre-treatment method, ultrasound-assisted surfactant-enhanced emulsification microextraction coupled with high performance liquid chromatography–diode array detector/electrospray ionisation mass spectrometry, was developed to determine diethofencarb and pyrimethanil residues in water and fruit juice samples. Tween 80 was used as an emulsifier and carbon tetrachloride was chosen as the extraction solvent, and no dispersive organic solvent was needed, which is typically required in common dispersive liquid–liquid microextraction methods. Several variables, such as the type and volume of extraction solvent and surfactant, extraction temperature and ultrasound extraction time were investigated and optimised. Under optimal conditions, the enrichment factors were 265 and 253 for diethofencarb and pyrimethanil, respectively. The limits of detection (LODs), calculated as three times the signal-to-noise ratio (S/N), were 0.01 μg L⁻¹ for both diethofencarb and pyrimethanil. The linearity of the method was obtained in the range of 0.05–2000 μg L⁻¹, with correlation coefficients of 0.9994–0.9998. The water (at fortified levels of 0.1 and 1.0 μg L⁻¹) and fruit juice samples (at fortified levels of 0.1 and 1.0 μg L⁻¹) were successfully analysed using the proposed method, and the relative recoveries were in the range of 88–114%, 93–111%, 86–117% and 94–101%, respectively.     

A novel method for the determination of total 1,3-octanediols in apple juice via 1,3-dioxanes by solid-phase microextraction and high-speed gas chromatography

In this work, a novel, simple and fast method based on solid-phase microextraction (SPME) followed by high-speed gas chromatography (HSGC) was developed for the analysis of total 1,3-octanediols in apple juices by means of derivatization reaction to volatile 1,3-dioxanes. The derivatization reaction, SPME conditions, glycosidically bound fraction and 1,3-nonanediol as a surrogate standard were studied. The formation of 1,3-dioxanes from 1,3-diols was confirmed by GC–MS. The method was validated obtaining a regression coefficient (r²) of 0.9996, precisions between 0.3 and 9.8%, extraction recoveries in the range 94.7–112.2% and LOD of 2.9 μg l⁻¹. Experimental design has been employed in the optimization of extraction factors and robustness assessment. The method was applied to the analysis of 21 Asturian apple varieties finding a double reciprocal relationship between the concentrations of saturated and unsaturated 1,3-octanediol.     

Characterization of calendula flower, milk-thistle fruit, and passion flower tinctures by HPLC-DAD and HPLC-MS

Summary As a part of an investigation of the content of herbal drug preparations and herbal medicinal products, we have investigated tinctures (prepared with 40:60 and 60:40 (%,v/v) ethanolwater) of calendular flower, milk-thistle fruit, and passion flower, which are, respectively, widely used for their anti-inflammatory properties, to treat hepatic injuries, and to treat tension and difficulty falling asleep. The aim of this work was to evaluate the flavonoid content, because flavonoids are the active constituents or markers of these herbal drugs, and to establish the best solvent for the extraction of the constituents. The findings reported herein both confirm the presence of several flavonoids previously identified in these herbal drugs and report the presence of others not previously described and identified here for the first time. In general the flavonol content was highest in 60% tinctures. A rapid, reversed-phase (RP) HPLC assay was developed and validated and enabled good separation of all classes of flavonoids including flavones, flavonols, flavanonols, and flavanolignans. Aglycones and mono, di, and triglycosides (bothO-andC-derivatives) of the flavonoids are also easily and satisfactorily separated. This method is thus proposed for the analysis of other herbal drugs or herbal drug preparations in which flavonoids are the active constituents or markers.