Patrizia Foglia

Identification of changes in Triticum durum L. leaf proteome in response to salt stress by two-dimensional electrophoresis and MALDI-TOF mass spectrometry

In order to understand the molecular basis of salt stress response, a proteomic approach, employing two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), was used to identify proteins affected by salinity in wheat (Triticum durum ‘Ofanto’). Identification of proteins, whose levels were altered, was performed by comparing protein patterns of salt-treated and control plants. A set of control plants was grown without NaCl addition under the same conditions as the salt-treated plants. Proteins were extracted from the leaves of untreated and NaCl-treated plants, and resolved using 24-cm immobilized pH gradient strips with a pH 4–7 linear gradient in the first dimension and a 12.5% sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension; the gels were stained with Coomassie and image analysis was performed. Quantitative evaluation, statistical analyses and MALDI-TOF MS characterization of the resolved spots in treated and untreated samples enabled us to identify 38 proteins whose levels were altered in response to salt stress. In particular, ten proteins were downregulated and 28 were upregulated. A possible role of these proteins in response to salinity is discussed.

Multiclass mycotoxin analysis in food, environmental and biological matrices with chromatography/mass spectrometry.

Mold metabolites that can elicit deleterious effects on other organisms are classified as mycotoxins. Human exposure to mycotoxins occurs mostly through the intake of contaminated agricultural products or residues due to carry over or metabolite products in foods of animal origin such as milk and eggs, but can also occur by dermal contact and inhalation. Mycotoxins contained in moldy foods, but also in damp interiors, can cause diseases in humans and animals. Nephropathy, various types of cancer, alimentary toxic aleukia, hepatic diseases, various hemorrhagic syndromes, and immune and neurological disorders are the most common diseases that can be related to mycotoxicosis. The absence or presence of mold infestation and its propagation are seldom correlated with mycotoxin presence. Mycotoxins must be determined directly, and suitable analytical methods are necessary. Hundreds of mycotoxins have been recognized, but only for a few of them, and in a restricted number of utilities, a maximum acceptable level has been regulated by law. However, mycotoxins seldom develop alone; more often various types and/or classes form in the same substrate. The co-occurrence might render the individual mycotoxin tolerance dose irrelevant, and therefore the mere presence of multiple mycotoxins should be considered a risk factor. The advantage of chromatography/mass spectrometry (MS) is that many compounds can be determined and confirmed in one analysis. This review illustrates the state-of-the-art of mycotoxin MS-based analytical methods for multiclass, multianalyte determination in all the matrices in which they appear. A chapter is devoted to the history of the long-standing coexistence and interaction among humans, domestic animals and mycotoxicosis, and the history of the discovery of mycotoxins. Quality assurance, although this topic relates to analytical chemistry in general, has been also examined for mycotoxin analysis as a preliminary to the systematic literature excursus. Sample handling is a crucial step to devise a multiclass analytical method; so when possible, it has been treated separately for a better comparison before tackling the instrumental part of the whole analytical method. This structure has resulted sometimes in unavoidable redundancies, because it was also important to underline the interconnection. Most reviews do not deal with all the possible mycotoxin sources, including the environmental ones. The focus of this review is the analytical methods based on MS for multimycotoxin class determination. Because the final purpose to devise multimycotoxin analysis should be the assessment of the danger to health of exposition to multitoxicants of natural origin (and possibly also the interaction with anthropogenic contaminants), therefore also the analytical methods for environmental relevant mycotoxins have been thoroughly reviewed. Finally, because the best way to shed light on actual risk assessment could be the individuation of exposure biomarkers, the review covers also the scarce literature on biological fluids.

Rapid-resolution liquid chromatography/mass spectrometry for determination and quantitation of polyphenols in grape berries.

A rapid-resolution liquid chromatography/mass spectrometric (RRLC/MS) method for detection and quantitation of polyphenols in grape berry skins and seeds has been developed. Pulp-free berry skins were treated with liquid nitrogen and ground; seeds were also ground. Then, 3 g of samples were extracted with 30 mL of a mixture of methanol/water/formic acid 70:30:1 (v/v/v) under sonication and 1 microL of the final extract was injected into two 100 x 2.1 mm i.d., 1.8 microm Zorbax Eclipse plus C18 columns connected in series. Compounds were fractionated using a gradient elution of acidified acetonitrile/methanol 50:50 (v/v)/water. Columns were thermostatted at 70 degrees C. MS was carried out on an Agilent 6410 QqQ instrument equipped with an electrospray ionization source. Positive and negative MS/MS product ion scans were used for compound identification, whereas positive full scan MS in the m/z range 200-1400 was used for quantitation. By means of mass spectra comparison, various flavonols, flavan-3-ols, anthocyanins and stilbenes were identified. Quantitation was performed by external calibration, and concentration values were corrected for matrix effect that was evaluated in separate experiments. Semi-quantitative estimation was performed for compounds for which standards were not commercially available. Recoveries ranged from 90-102% with relative standard deviation (RSD) <5%, whereas the between samples RSD was in the range 4-12%. Two surrogate standards were used for quality control. The developed method was applied to analyze the polyphenol content of three Vitis vinifera table cultivars at physiological maturity and after proper preservation for 6 weeks. Results demonstrated that during preservation about half of the polyphenol content was lost.

Flavonoids: chemical properties and analytical methodologies of identification and quantitation in foods and plants

Flavonoids have been recognised as one of the largest and most widespread groups of plant secondary metabolites, with marked antioxidant properties. The general name flavonoid refers to a class of more than 6500 molecules based upon a 15-carbon skeleton. In this paper a general overview of flavonoids, their classification, structures and analytical methods for their determination is presented.

Evolution of the protein corona of lipid gene vectors as a function of plasma concentration.

The concept that the effective unit of interest in the cell-nanomaterial interaction is the particle and its corona of associated proteins is emerging. Here we investigate the compositional evolution of the protein corona of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) cationic liposomes (CLs) and DOTAP/DNA lipoplexes over a wide range of plasma concentrations (2.5-80%). The composition of the hard corona of lipoplexes is quite stable, but that of CLs does evolve considerably. We show that the protein corona of CLs is made of both low-affinity and competitive-binding proteins whose relative abundance changes with the plasma concentration. This result may have deep biological implications for the application of lipid-based gene vectors both in vitro and in vivo.

Intact protein separation by chromatographic and/or electrophoretic techniques for top-down proteomics

Mass spectrometry used in combination with a wide variety of separation methods is the principal methodology for proteomics. In bottom-up approach, proteins are cleaved with a specific proteolytic enzyme, followed by peptide separation and MS identification. In top-down approach intact proteins are introduced into the mass spectrometer. The ions generated by electrospray ionization are then subjected to gas-phase separation, fragmentation, fragment separation, and automated interpretation of mass spectrometric and chromatographic data yielding both the molecular weight of the intact protein and the protein fragmentation pattern. This approach requires high accuracy mass measurement analysers capable of separating the multi-charged isotopic cluster of proteins, such as hybrid ion trap-Fourier transform instruments (LTQ-FTICR, LTQ-Orbitrap). Front-end separation technologies tailored for proteins are of primary importance to implement top-down proteomics. This review intends to provide the state of art of protein chromatographic and electrophoretic separation methods suitable for MS coupling, and to illustrate both monodimensional and multidimensional approaches used for LC-MS top-down proteomics. In addition, some recent progresses in protein chromatography that may provide an alternative to those currently employed are also discussed.

Development and validation of a liquid chromatography/atmospheric pressure photoionization-tandem mass spectrometric method for the analysis of mycotoxins subjected to commission regulation (EC) No. 1881/2006 in cereals.

A sensitive and reliable liquid chromatography/photoionization (APPI) tandem mass spectrometry method has been developed for determining nine selected mycotoxins in wheat and maize samples. The analytes were chosen on the basis of the mycotoxins under EU Commission Regulation (EC) No. 1881/2006, i.e., deoxynivalenol (DON), zearalenone (ZON), aflatoxins (AFs), and ochratoxin A (OTA), and considering the possibility of a near future regulation for T-2 and HT-2 toxins. Mycotoxins were extracted from samples by means of an one-step solvent extraction without any cleanup. The developed multi-mycotoxin method permits simultaneous, simple, and rapid determination of several co-existing toxins separated in a single chromatographic run, in which AFs, T-2 and HT-2 toxin are acquired in positive, while OTA, DON and ZON in negative mode. Although a moderate signal suppression was noticeable, matrix effect did not give significant differences at p=0.05. Then, calibration in standard solution were used for quantitation. Based on the EU Commission Decision 2002/657/EC, the method was in-house validated in terms of ruggedness, specificity, linearity, trueness, within-laboratory reproducibility, decision limit (CCalpha) and detection capability (CCbeta). For all the analytes, the regression coefficient r ranged between 0.8752 (DON in wheat) and 0.9465 (ZON in maize), biases related to mean concentrations were from -13% to +12% of the nominal spiking level, and the overall within-laboratory reproducibility ranged 3-16%; finally, CCalpha values did not differ more than 20% and CCbeta not more than 42% from their respective maximum limit. Method quantification limits ranged from 1/20 (AFG1) to 1/4 (AFG2 and OTA) the maximum limit established by European Union in the Commission Regulation (EC) No. 1881/2006 and its subsequent amendments.

Multiclass analysis of mycotoxins in biscuits by high performance liquid chromatography-tandem mass spectrometry. Comparison of different extraction procedures.

A sensitive, simple and rapid method for the simultaneous determination of 19 mycotoxins in biscuits (a dry matrix containing cereals and egg) has been developed using high performance liquid chromatography coupled to tandem mass spectrometry with electrospray source working in both positive and negative mode. Due to the matrix complexity and the high amount of contaminants, a solid phase extraction method using graphitized carbon black was optimized for an effective clean-up step. Accuracy was carried out in the selected matrix using blank samples spiked at three analyte concentrations. Recoveries between 63 and 107% and relative standard deviations lower than 12% were obtained. For all considered mycotoxin classes, i.e. thricotecenes A and B, zearalenone and its metabolites, fumonisins, ochratoxin A, enniatins and their structurally related beauvericin, the method was validated in terms of linearity, recovery, matrix effect, precision, limit of detection and limit of quantification. Matrix-matched calibration was used for quantification purposes, in order to compensate for matrix effect. The coefficients of determination obtained were in the range of 0.9927-1. The limits of quantification, ranging from 0.04μgkg(-1) for enniatin B1 to 80.2μgkg(-1) for nivalenol, were always lower than maximum permitted levels for every regulated mycotoxin by the current European legislation.

Comparison of extraction methods for the identification and quantification of polyphenols in virgin olive oil by ultra-HPLC-QToF mass spectrometry

In this work, liquid-liquid extraction (LLE) and solid phase extraction (SPE), of polyphenols from a VOO sample were optimised by a Plackett-Burman experimental design; then the two extraction techniques capabilities were compared. By using HPLC-DAD, the extraction ability of SPE with the diol phase and LLE were similar. The two methods were further evaluated with ultra HPLC-ESI QToF in negative ion mode by recoveries of standards and matched comparison of the peak area of 40 identified and 27 unidentified compounds. Conclusions indicate that LLE gives better recoveries for highly polar, non-polar, and some polyphenols suspected to contain a nitrogen atom, while for the others the two methods seem to be equally suitable. The presence of nitrogen-containing polyphenols was confirmed in positive ionisation mode in LLE extract, whereas in the SPE extract they were not present. One of them was tentatively identified as a compound containing tyrosine and methyl-decarboxymetyl-eleanoic acid moieties.

DNA affects the composition of lipoplex protein corona: a proteomics approach.

The distribution of drug delivery systems into the body is affected by plasma proteins adsorbed onto their surface. Furthermore, an exact understanding of the structure and morphology of drug carriers is fundamental to understand their role as gene delivery systems. In this work, the adsorption of human plasma proteins bound to cationic liposomes and to their relative DNA lipoplexes was compared. A shotgun proteomics approach based on HPLC coupled to high resolution MS was used for an efficient identification of proteins adsorbed onto liposome and lipoplex surfaces. The distinct pattern of proteins adsorbed helps to better understand the DNA compaction process. The experimental evidence leads us to hypothesize that polyanionic DNA is associated to the lipoplex surface and can interact with basic plasma proteins. Such a finding is in agreement with recent results showing that lipoplexes are multilamellar DNA/lipid domains partially decorated with DNA at their surface. Proteomics experiments showed that the lipoplex corona is rich of biologically relevant proteins such as fibronectin, histones and complement proteins. Our results provide novel insights to understand how lipoplexes activate the immune system and why they are rapidly cleared from the blood stream. The differences in the protein adsorption data detected in the presented experiments could be the basis for the establishment of a correlation between protein adsorption pattern and in vivo fate of intravenously administered nanoparticles and will require some consideration in the future.