Development of an enrichment method for endogenous phosphopeptide characterization in human serum.

The work describes the development of an enrichment method for the analysis of endogenous phosphopeptides in serum. Endogenous peptides can play significant biological roles, and some of them could be exploited as future biomarkers. In this context, blood is one of the most useful biofluids for screening, but a systematic investigation of the endogenous peptides, especially phosphorylated ones, is still lacking, mainly due to the lack of suitable analytical methods. Thus, in this paper, different phosphopeptide enrichment strategies were pursued, based either on metal oxide affinity chromatography (MOAC, in the form of commercial TiO2 spin columns or magnetic graphitized carbon black-TiO2 composite), or on immobilized metal ion affinity chromatography (IMAC, in the form of Ti4+-IMAC magnetic material or commercial Fe3+-IMAC spin columns). While MOAC strategies proved completely unsuccessful, probably due to interfering phospholipids displacing phosphopeptides, the IMAC materials performed very well. Different sample preparation strategies were tested, comprising direct dilution with the loading buffer, organic solvent precipitation, and lipid removal from the matrix, as well as the addition of phosphatase inhibitors during sample handling for maximized endogenous phosphopeptide enrichment. All data were acquired by a shotgun peptidomics approach, in which peptide samples were separated by reversed-phase nanoHPLC hyphenated with high-resolution tandem mass spectrometry. The devised method allowed the identification of 176 endogenous phosphopeptides in fresh serum added with inhibitors by the direct dilution protocol and the Ti4+-IMAC magnetic material enrichment, but good results could also be obtained from the commercial Fe3+-IMAC spin column adapted to the batch enrichment protocol.

Evaluation of column length and particle size effect on the untargeted profiling of a phytochemical mixture by using UHPLC coupled to high-resolution mass spectrometry.

Liquid chromatography coupled to high-resolution mass spectrometry is the technique of choice for the untargeted profiling of food matrices. Despite the high potential of high-resolution mass spectrometry, when dealing with complex mixtures, an efficient separation technique is also needed. The novel core-shell chromatographic columns packed with sub-2 µm sized particles are claimed to show very good resolution. However, the analytes retention can be significantly altered when working under ultra-high performance chromatographic conditions. In this work, an evaluation of four chromatographic systems, with either a single or two in-series Kinetex™ C18 columns, either packed with 2.6 or 1.7 µm particles, is presented for the targeted analysis of a standard mixture and the untargeted analysis of a strawberry extract. An ultra-high performance chromatographic system coupled via an electrospray source to a hybrid quadrupole-Orbitrap mass spectrometer was used. From the extensive comparison, a surprising result was obtained, namely, that the system identifying the largest number of features was the one with two in-series connected columns with the larger particle size. The inconsistency among the theoretical assumptions and the applicative findings points out the importance of an extensive chromatographic evaluation for the comprehensive untargeted profiling of complex real samples.

New Magnetic Graphitized Carbon Black TiO2 Composite for Phosphopeptide Selective Enrichment in Shotgun Phosphoproteomics.

Graphitized carbon black (GCB) has been employed for extraction of several classes of analytes, due to the large surface area and the unique chemistry of its surface groups that allows for extracting a wide range of analytes, including polar, acidic compounds. Despite the fact that structurally related materials, such as graphene, found application as hybrid-components in phosphoproteomics, surprisingly, GCB has never been used for the selective enrichment of phosphopeptides. For this purpose, in the present work we used GCB to prepare a magnetic composite with TiO2 (mGCB@TiO2) that was then applied to yeast total extracts. We exploited the high surface area provided by nanostructures, the presence of nano-TiO2 for selective binding of phosphopeptides, and the magnetic responsiveness of magnetite for solid-phase separation. The material was extensively characterized at each modification step by transmission electron microscopy, Fourier-transformed infrared spectroscopy, thermogravimetric analysis, Raman spectroscopy, and porosimetry. Next, the new system was applied for the enrichment of casein phosphopeptides from a simulated tryptic digest with bovine serum albumin (BSA:casein, 100:1). Finally, after assessing the potential applicability, the composite was employed for enriching phosphopeptides from yeast protein digests. This allowed us not only to optimize the enrichment protocol but also to fully compare its performance to commercial TiO2 spin columns. To achieve this aim, the optimized enrichment protocol was included in a typical shotgun proteomics analytical workflow based on nanoHPLC-MS/MS analysis.

An ICP-MS-Based Analytical Strategy for Assessing Compliance with the Ban of E 171 as a Food Additive on the EU Market.

A method was developed for the determination of total titanium in food and food supplements by inductively coupled plasma mass spectrometry (ICP-MS) after microwave-assisted acid digestion of samples. Five food supplements, including one certified reference material, and 15 food products were used for method development. Key factors affecting the analytical results, such as the composition of the acid mixture for sample digestion and the bias from spectral interferences on the different titanium isotopes, were investigated. Resolution of interferences was achieved by ICP-MS/MS with ammonia adduct formation and viable conditions for control laboratories equipped with standard quadrupole instruments were identified. The method was successfully validated and enables rapid screening of samples subject to confirmatory analysis for the presence of TiO2 particles. For the latter, single-particle ICP-MS (spICP-MS) analysis after chemical extraction of the particles was used. The two methods establish a viable analytical strategy for assessing the absence of titania particles in food products on the EU market following the E 171 ban as a food additive.

Agglomeration Behavior and Fate of Food-Grade Titanium Dioxide in Human Gastrointestinal Digestion and in the Lysosomal Environment.

In the present study, we addressed the knowledge gaps regarding the agglomeration behavior and fate of food-grade titanium dioxide (E 171) in human gastrointestinal digestion (GID). After thorough multi-technique physicochemical characterization including TEM, single-particle ICP-MS (spICP-MS), CLS, VSSA determination and ELS, the GI fate of E 171 was studied by applying the in vitro GID approach established for the regulatory risk assessment of nanomaterials in Europe, using a standardized international protocol. GI fate was investigated in fasted conditions, relevant to E 171 use in food supplements and medicines, and in fed conditions, with both a model food and E 171-containing food samples. TiO2 constituent particles were resistant to GI dissolution, and thus, their stability in lysosomal fluid was investigated. The biopersistence of the material in lysosomal fluid highlighted its potential for bioaccumulation. For characterizing the agglomeration degree in the small intestinal phase, spICP-MS represented an ideal analytical tool to overcome the limitations of earlier studies. We demonstrated that, after simulated GID, in the small intestine, E 171 (at concentrations reflecting human exposure) is present with a dispersion degree similar to that obtained when dispersing the material in water by means of high-energy sonication (i.e., ≥70% of particles <250 nm).

Short- and Long-Term Effects of Suboptimal Selenium Intake and Developmental Lead Exposure on Behavior and Hippocampal Glutamate Receptors in a Rat Model.

Selenium (Se) is an essential trace element required for normal development as well as to counteract the adverse effects of environmental stressors. Conditions of low Se intake are present in some European countries. Our aim was to investigate the short- and long-term effects of early-life low Se supply on behavior and synaptic plasticity with a focus on the hippocampus, considering both suboptimal Se intake per se and its interaction with developmental exposure to lead (Pb). We established an animal model of Se restriction and low Pb exposure; female rats fed with an optimal (0.15 mg/kg) or suboptimal (0.04 mg/kg) Se diet were exposed from one month pre-mating until the end of lactation to 12.5 µg/mL Pb via drinking water. In rat offspring, the assessment of motor, emotional, and cognitive endpoints at different life stages were complemented by the evaluation of the expression and synaptic distribution of NMDA and AMPA receptor subunits at post-natal day (PND) 23 and 70 in the hippocampus. Suboptimal Se intake delayed the achievement of developmental milestones and induced early and long-term alterations in motor and emotional abilities. Behavioral alterations were mirrored by a drop in the expression of the majority of NMDA and AMPA receptor subunits analyzed at PND 23. The suboptimal Se status co-occurring with Pb exposure induced a transient body weight increase and persistent anxiety-like behavior. From the molecular point of view, we observed hippocampal alterations in NMDA (Glun2B and GluN1) and AMPA receptor subunit trafficking to the post-synapse in male rats only. Our study provides evidence of potential Se interactions with Pb in the developing brain.

Determination of the Transport Efficiency in spICP-MS Analysis Using Conventional Sample Introduction Systems: An Interlaboratory Comparison Study.

In single particle inductively coupled plasma mass spectrometry (spICP-MS), the transport efficiency is fundamental for the correct determination of both particle number concentration and size. In the present study, transport efficiency was systematically determined on three different days with six carefully characterised gold nanoparticle (AuNP) suspensions and in seven European and US expert laboratories using different ICP-MS instruments and spICP-MS software. Both particle size-(TES)-and particle frequency-(TEF)-methods were applied. The resulting transport efficiencies did not deviate much under ideal conditions. The TEF method however systematically resulted in lower transport efficiencies. The extent of this difference (0-300% rel. difference) depended largely on the choice and storage conditions of the nanoparticle suspensions used for the determination. The TES method is recommended when the principal measurement objective is particle size. If the main aim of the measurement is the determination of the particle number concentration, the TEF approach could be preferred as it might better account for particle losses in the sample introduction system.

Essential and toxic elements in sustainable and underutilized seafood species and derived semi-industrial ready-to-eat products.

Blue whiting (Micromesistius poutassou), pouting (Trisopterus luscus) and common dab (Limanda limanda) are underutilized fish species attractive in terms of sustainability. However, there is limited information about their nutritional characteristics as well as potential presence of environmental contaminants. Specimens caught in European waters were characterised for their content of essential and toxic elements. The three species, along with common carp and blue mussels, relevant for sustainable production too, were employed as raw materials for the development of semi-industrial ready-to-eat products. Calcium, copper, iodine, iron, selenium, zinc, cadmium, lead, mercury, and nickel were determined by ICP-MS, whereas methylmercury was determined by HPLC-ICP-MS. These two techniques were also used to determine arsenic and inorganic arsenic, respectively, in blue mussel and derived products. Differences in element contents were related to the biology and ecology of the examined species. Intake of nutrients and exposure to contaminants were assessed in relation to the relevant DRVs and HBGVs, respectively. All the species were found to be valuable dietary sources of selenium. Pouting was rich in iodine and mussels were good sources of iodine and iron. These two species had comparatively higher levels of mercury and lead, respectively. However, the levels of contaminants were generally of no concern in both raw materials and products. Iodine bioaccessibility was studied in blue whiting, a species with an intermediate iodine content, and found to be 98%. Selenium:mercury molar ratios were assessed and found to be favourable. The semi-industrial products were found to be good sources of selenium and many of them provided appreciable amounts of calcium, iron, copper and zinc.

Semi-industrial development of nutritious and healthy seafood dishes from sustainable species.

This study aimed to devise innovative, tailor-made, appealing, tasty and semi-industrialized dishes, using sustainable and under-utilized seafood species (bib, common dab, common carp, blue mussel and blue whiting), that can meet the specific nutritional and functional needs of children (8-10-years), pregnant women (20-40-years) and seniors (≥60-years). Hence, contests were organised among cooking schools from 6 European countries and the best recipes/dishes were reformulated, semi-industrially produced and chemically and microbiologically evaluated. The dishes intended for: (i) children and pregnant women had EPA + DHA and I levels that reached the target quantities, supporting the claim as "high in I"; and (ii) seniors were "high in protein" (24.8%-Soup_S and 34.0%-Balls_S of the energy was provided by proteins), "high in vitamin B12", and had Na contents (≤0.4%) below the defined limit. All dishes reached the vitamin D target value. Sausages_C, Roulade_P, Fillet_P and Balls_S had a well-balanced protein/fat ratio. Roulade_P presented the highest n-3 PUFA/n-6 PUFA ratio (3.3), while Sausages_C the lowest SFA/UNS ratio (0.2). Dishes were considered safe based on different parameters (e.g. Hg-T, PBDEs, Escherichia coli). All represent dietary sources contributing to meet the reference intakes of target nutrients (33->100%), providing valuable options to overcome nutritional and functional imbalances of the three groups.

Dietary exposure of the Italian population to nickel: The national Total Diet Study.

Dietary exposure of the Italian population to nickel has been assessed in the national Total Diet Study (TDS). Occurrence data were combined with national individual consumption data to estimate mean and high level dietary exposure of population subgroups according to age and gender, both at the national level and for the four main geographical areas of Italy. The mean chronic dietary exposure of infants and toddlers, children, adolescents, adults, and the elderly were 4.00, 4.57, 2.57, 1.55, and 1.47 µg/kg bw per day, respectively. These intakes lie in the intermediate range of exposure estimates from TDS carried out in other countries. Main contributors to the total nickel exposure for children and adolescents were 'sweet products' and 'cereals and cereal products'. In adults and the elderly nearly 30% of the exposure was associated to the consumption of 'cereals and cereal products'. Mean and 95th percentile chronic dietary exposure was below the TDI in all age groups. For the risk characterisation of acute oral exposure, exposure data for consumers only in the adult population were compared with the reference point for systemic contact dermatitis. Consumption of 'cocoa', 'chocolate', 'crustaceans and molluscs', 'pulses' had remarkable potential to elicit adverse effects in nickel-sensitised individuals.

Saliva as a source of new phosphopeptide biomarkers: Development of a comprehensive analytical method based on shotgun peptidomics.

The paper describes the development of an enrichment method for the analysis of the endogenous phosphopeptides in saliva. The method was based on magnetic solid phase extraction by a magnetic graphitized carbon black-TiO2 composite material and was developed considering different saliva pre-treatments, namely C18 solid phase extraction for purification, direct dilution in loading buffer or acetonitrile precipitation. The method was based on a shotgun proteomics workflow and the enriched peptide mixture was analysed by nanoHPLC and high resolution tandem mass spectrometry. Acetonitrile precipitation provided the best results, with up to 165 endogenous phosphopeptides identified in saliva samples from healthy individuals. The physico-chemical features of the identified endogenous phosphopeptides indicated that such peptides were large, hydrophilic and basic.

New Ti-IMAC magnetic polymeric nanoparticles for phosphopeptide enrichment from complex real samples.

The work describes the preparation of a new magnetic phase for batch enrichment of phosphopeptides. The material exploits the advantages of magnetic solid phase extraction and couples them with the most employed approach for phosphopeptide enrichment, i.e. Ti4+-IMAC. In order to immobilize Ti4+ ions on the surface of the magnetite nanoparticles, they were first covered by a silica shell and then modified to expose at the surface bromine containing groups. Glycidyl methacrylate was subsequently polymerized from these groups using the "grafting from" approach by the activator regenerated by electron transfer-atom transfer radical polymerization (ARGET-ATRP) technique. Finally, the glycidyl groups were reacted with iminodiacetic acid to functionalize the material with moieties suitable for coordination. The prepared material was extensively characterized and subsequently tested for enrichment of a bovine serum albumin mixture with casein to ascertain its potential. With positive results, the new magnetic polymeric material was further employed to set up an enrichment method on yeast protein digest based on shotgun proteomics. The sample to phase ratio was optimized and the best condition compared to a commercial TiO2 spin column. At the end of the comparison, the new material proved better and could enrich a larger total number of phosphopeptides with increased selectivity. All these conclusions and the test performed on a real complex sample within the final shotgun application further support the applicability of the new material in phosphopeptide analysis of real matrices.

Multiresidue analysis of endocrine-disrupting compounds and perfluorinated sulfates and carboxylic acids in sediments by ultra-high-performance liquid chromatography-tandem mass spectrometry.

A multiresidue analytical method for the determination of 11 perfluorinated compounds and 22 endocrine-disrupting compounds (ECDs) including 13 natural and synthetic estrogens (free and conjugated forms), 2 alkylphenols, 1 plasticiser, 2 UV-filters, 1 antimicrobial, and 2 organophosphorus compounds in sediments has been developed. Ultrasound-assisted extraction followed by solid phase extraction (SPE) with graphitized carbon black (GCB) cartridge as clean-up step were used. The extraction process yield was optimized in terms of solvent composition. Then, a 3(2) experimental design was used to optimize solvent volume and sonication time by response surface methodology, which simplifies the optimization procedure. The final extract was analyzed by ultra-high performance liquid chromatography coupled with tandem mass spectrometry. The optimized sample preparation method is simple and robust, and allows recovery of ECDs belonging to different classes in a complex matrix such as sediment. The use of GCB for SPE allowed to obtain with a single clean-up procedure excellent recoveries ranging between 75 and 110% (relative standard deviation <16%). The developed methodology has been successfully applied to the analysis of ECDs in sediments from different rivers and lakes of the Lazio Region (Italy). These analyses have shown the ubiquitous presence of chloro-substituted organophosphorus flame retardants and bisphenol A, while other analyzed compounds were occasionally found at concentration between the limit of detection and quantification.

Phosphopeptide enrichment: Development of magnetic solid phase extraction method based on polydopamine coating and Ti(4+)-IMAC.

Protein post translational modifications currently represent one of the main challenges with proteomic analysis, due to the important biological role they play within cells. Protein phosphorylation is one of the most important, with several approaches developed for phosphopeptides enrichment and analysis, essential for comprehensive phosphoproteomic analysis. However, the development of new materials for phosphopeptides enrichment may overcome previous drawbacks and improve enrichment of such peptides. In this regard, new magnetic stationary phases based on polydopamine coating and Ti(4+) immobilization exploit the potential of IMAC enrichment and couple it with the versatility of magnetic solid phase extraction. In this work the use of such stationary phase was extended from the MALDI proof of concept stage with the development of an optimized method for phosphopeptides enrichment compatible with typical shotgun proteomics experimental workflows. Different loading and elution buffers were tested to improve phosphopeptides recovery and enrichment selectivity. Finally, the analysis of isolated peptides pointed out that polydopamine alone is an ideal support matrix for polar post translational modifications because it enables to reduce unspecific binding and preferentially binds hydrophilic peptides.