Serena Stampachiacchiere

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.

Advances in liquid chromatography-high-resolution mass spectrometry for quantitative and qualitative environmental analysis.

This review summarizes the advances in environmental analysis by liquid chromatography–high-resolution mass spectrometry (LC–HRMS) during the last decade and discusses different aspects of their application. LC–HRMS has become a powerful tool for simultaneous quantitative and qualitative analysis of organic pollutants, enabling their quantitation and the search for metabolites and transformation products or the detection of unknown compounds. LC–HRMS provides more information than low-resolution (LR) MS for each sample because it can accurately determine the mass of the molecular ion and its fragment ions if it can be used for MS–MS. Another advantage is that the data can be processed using either target analysis, suspect screening, retrospective analysis, or non-target screening. With the growing popularity and acceptance of HRMS analysis, current guidelines for compound confirmation need to be revised for quantitative and qualitative purposes. Furthermore, new commercial software and user-built libraries are required to mine data in an efficient and comprehensive way. The scope of this critical review is not to provide a comprehensive overview of the many studies performed with LC–HRMS in the field of environmental analysis, but to reveal its advantages and limitations using different workflows.

Comparative analysis of metabolic proteome variation in ascorbate-primed and unprimed wheat seeds during germination under salt stress

UNLABELLED Seed priming with ascorbic acid improves salt tolerance in durum wheat. For understanding the potential mechanisms underlying this priming effect a gel-free shotgun proteomic analysis was performed comparing unprimed to ascorbate-primed wheat seed during germination under saline and non-saline conditions. Since seed germination is the result of interplay or cross-talk between embryo and embryo-surrounding tissues, we studied the variation of metabolic proteome in both tissues separately. 167 of 697 identified and 69 of 471 identified proteins increase or decrease in abundance significantly in response to priming and/or salinity compared to untreated, unstressed control in embryo and embryo-surrounding tissues, respectively. In untreated wheat embryo salt stress was accompanied by change in 129 proteins, most of which are belonging to metabolism, energy, disease/defense, protein destination and storage categories. Ascorbate pretreatment prevents and counteracts the effects of salinity upon most of these proteins and changes specifically the abundance of 35 others proteins, most of which are involved in metabolism, protein destination and storage categories. Hierarchical clustering analysis revealed three and two major clusters of protein expression in embryo and embryo-surrounding tissues, respectively. This study opens promising new avenues to understand priming-induced salt tolerance in plants. BIOLOGICAL SIGNIFICANCE To clearly understand how ascorbate-priming enhance the salt tolerance of durum wheat during germination, we performed for the first time a comparative shotgun proteomic analysis between unprimed and ascorbate-primed wheat seeds during germination under saline and non-saline conditions. Furthermore, since seed germination is the result of interplay or cross-talk between embryo and embryo-surrounding tissues we analyzed the variation of metabolic proteome in both tissues separately. 1168 proteins exhibiting greater molecular weight diversity (ranging from 5 to 258kDa) were identified. Among them, 167 and 69 proteins were increased or decreased in abundance significantly by priming and/or salinity as compared to control, in embryo and embryo-surrounding tissues respectively. Ascorbate pretreatment alleviates the effects of salinity upon most of these proteins, particularly those involved in metabolism, energy, disease/defense, protein destination and storage functions. Hierarchical clustering analysis revealed three and two major clusters of protein accumulation in embryo and embryo-surrounding tissues, respectively. These results may provide new avenues for understanding and advancing priming-induced salt tolerance in crop plants.

Heterosis profile of sunflower leaves: A label free proteomics approach

UNLABELLED Heterosis is the superior performance of heterozygous F1-hybrid plants compared with their homozygous genetically distinct parents. The proteome of leaves of one sunflower hybrid and its parental inbred lines was analyzed by label free LC-MS/MS. A total of 1998 proteins were identified. Among them 38 proteins indicated heterosis pattern in hybrid compared with midparents. The results showed an increment of photosynthesis capacity, assimilation rate, nitrogen fixation, cell growth and reducing in some energy-consuming processes like protein production, response to stresses and respiration. These results suggest that heterosis mechanisms increase input energy of plant with reinforcement of carbon fixation pathway and reduction in consumed energy toward production of superior hybrid. This study could help to better elucidate what mechanisms are involved in heterosis of sunflower leaves and what happens at proteome level. BIOLOGICAL SIGNIFICANCE The current work describes the first study in which gel-free shotgun proteomics was used to compare the proteome of leaves of one sunflower hybrid to its parental inbred lines. In this study 1998 proteins were identified from sunflower leaves with label free nano LC-MS/MS. The numbers of 38 proteins significantly showed heterosis pattern. The comparison between hybrid and parental inbred lines showed that hybrid vigor is actually linked by emphasizing the assimilation rate and low energy consumption.

Protein Profile of Mature Soybean Seeds and Prepared Soybean Milk

The soybean (Glycine max (L.) Merrill) is economically the most important bean in the world, providing a wide range of vegetable proteins. Soybean milk is a colloidal solution obtained as water extract from swelled and ground soybean seeds. Soybean proteins represent about 35-40% on a dry weight basis and they are receiving increasing attention with respect to their health effects. However, the soybean is a well-recognized allergenic food, and therefore, it is urgent to define its protein components responsible for the allergenicity in order to develop hypoallergenic soybean products for sensitive people. The main aim of this work was the characterization of seed and milk soybean proteome and their comparison in terms of protein content and specific proteins. Using a shotgun proteomics approach, 243 nonredundant proteins were identified in mature soybean seeds.

Simultaneous Determination of Naturally Occurring Estrogens and Mycoestrogens in Milk by Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry Analysis.

A simple, fast, and reproducible method for the simultaneous determination of natural estrogens and mycoestrogens (resorcylic acid lactones) in milk by ultrahigh-performance liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry (UHPLC/ESI-MS/MS) is described. The extraction was carried out by solid-phase extraction (SPE) using graphitized carbon black as solid sorbent. The use of carbon black allowed us to avoid any type of sample pretreatment, and the extraction was performed simply by diluting milk samples in water. Correlation coefficient values were obtained in the range between 0.9991 and 1, with good recoveries (67-107% at the lowest spiked level), repeatability (4.8-16.8%), and reproducibility (3.2-16.3%). Moreover, a very low matrix effect was observed for both estrogens and mycoestrogens. With respect to a previous method based on SPE with Oasis MAX cartridges, the one here described allowed us to detect all the analytes under investigation, at the lowest tested concentration level, including free estrogens (in particular estriol). Finally, the developed UHPLC/ESI-MS/MS method was applied to the analysis of some whole milk samples from different lactating animals (cow, goat, and donkey) as well as ultrahigh-temperature-treated cow milk and powder milk samples.

Characterization of quinoa seed proteome combining different protein precipitation techniques: Improvement of knowledge of nonmodel plant proteomics

A shotgun proteomics approach was used to characterize the quinoa seed proteome. To obtain comprehensive proteomic data from quinoa seeds three different precipitation procedures were employed: MeOH/CHCl3 /double-distilled H2 O, acetone either alone or with trichloroacetic acid; the isolated proteins were then in-solution digested and the resulting peptides were analyzed by nano-liquid chromatography coupled to tandem mass spectrometry. However, since quinoa is a nonmodel plant species, only a few protein sequences are included in the most widely known protein sequence databases. To improve the data reliability a UniProt subdatabase, containing only proteins of Caryophillales order, was used. A total of 352 proteins were identified and evaluated both from a qualitative and quantitative point of view. This combined approach is certainly useful to increase the final number of identifications, but no particular class of proteins was extracted and identified in spite of the different chemistries and the different precipitation protocols. However, with respect to the other two procedures, from the relative quantitative analysis, based on the number of spectral counts, the trichloroacetic acid/acetone protocol was the best procedure for sample handling and quantitative protein extraction. This study could pave the way to further high-throughput studies on Chenopodium Quinoa.

Multiresidue determination of UV filters in water samples by solid-phase extraction and liquid chromatography with tandem mass spectrometry analysis.

UV filters, contained in sunscreens and other cosmetic products, as well as in some plastics and industrial products, are nowadays considered contaminants of emerging concern because their widespread and increasing use has lead to their presence in the environment. Furthermore, some UV filters are suspected to have endocrine disruption activity. In the present work, we developed an analytical method based on liquid chromatography with tandem mass spectrometry for the determination of UV filters in tap and lake waters. Sixteen UV filters were extracted from water samples by solid-phase extraction employing graphitized carbon black as adsorbent material. Handling 200 mL of water sample, satisfactory recoveries were obtained for almost all the analytes. The limits of detection and quantification of the method were comparable to those reported in other works, and ranged between 0.7-3.5 and 1.9-11.8 ng/L, respectively; however in our case the number of investigated compounds was larger. The major encountered problem in method development was to identify the background contamination sources and reduce their contribution. UV filters were not detected in tap water samples, whereas the analyses conducted on samples collected from three different lakes showed that the swimming areas are most subject to UV filter contamination.

Proteomic platform for the identification of proteins in olive (Olea europaea) pulp

The nutritional and cancer-protective properties of the oil extracted mechanically from the ripe fruits of Olea europaea trees are attracting constantly more attention worldwide. The preparation of high-quality protein samples from plant tissues for proteomic analysis poses many challenging problems. In this study we employed a proteomic platform based on two different extraction methods, SDS and CHAPS based protocols, followed by two precipitation protocols, TCA/acetone and MeOH precipitation, in order to increase the final number of identified proteins. The use of advanced MS techniques in combination with the Swissprot and NCBI Viridiplantae databases and TAIR10 Arabidopsis database allowed us to identify 1265 proteins, of which 22 belong to O. europaea. The application of this proteomic platform for protein extraction and identification will be useful also for other proteomic studies on recalcitrant plant/fruit tissues.

Shotgun proteomic analysis of soybean embryonic axes during germination under salt stress.

Seed imbibition and radicle emergence are generally less affected by salinity in soybean than in other crop plants. In order to unveil the mechanisms underlying this remarkable salt tolerance of soybean at seed germination, a comparative label‐free shotgun proteomic analysis of embryonic axes exposed to salinity during germination sensu stricto (GSS) was conducted. The results revealed that the application of 100 and 200 mmol/L NaCl stress was accompanied by significant changes (>2‐fold, P<0.05) of 97 and 75 proteins, respectively. Most of these salt‐responsive proteins (70%) were classified into three major functional categories: disease/defense response, protein destination and storage and primary metabolism. The involvement of these proteins in salt tolerance of soybean was discussed, and some of them were suggested to be potential salt‐tolerant proteins. Furthermore, our results suggest that the cross‐protection against aldehydes, oxidative as well as osmotic stress, is the major adaptive response to salinity in soybean.