Proteomic study of pilocytic astrocytoma pediatric brain tumor intracystic fluid.

Liquid chromatography in coupling with high-resolution ESI-LTQ-Orbitrap mass spectrometry was applied for a proteomic study of pediatric pilocytic astrocytoma brain tumor intracystic fluid by an integrated top-down/bottom-up platform. Both of the proteomic strategies resulted complementary and support each other in contributing to a wide characterization of the protein and peptide content of the tumor fluid. Top-down approach allowed to identify several proteins and peptides involved in different biological activities together with the characterization of interesting proteoforms such as fibrinopeptide A and its truncated form, fibrinopeptide B, complement C3f fragments, ß-thymosin peptides, ubiquitin, several apolipoproteins belonging to A and C families, apolipoprotein J and D, and cystatin C. Of particular interest resulted the identification of a N-terminal truncated cystatin C proteoform, likely involved in immune response mechanism modulations and the identification of oxidized and glycosylated apolipoproteins including disulfide bridge dimeric forms. The bottom-up approach confirmed some of the experimental data findings together with adding the characterization of high-molecular-mass proteins in the samples. These data could contribute to elucidate the molecular mechanisms involved in onset and progression of the disease and cyst development.

Heavy metals and trace elements in milk and dairy products in the Lazio region (Central Italy).

A study was carried out to determine the concentration of heavy metals and trace elements in milk and dairy products collected from local farms, supermarkets, or food retailers in the region of Lazio (Central Italy). Persistent exposure to metal contamination is of particular concern for human health, as it can cause different serious disorders. The monitoring of the matrices studied is therefore important, given their high consumption in the daily diet. The elements determined by ICP-MS (Inductively Coupled Plasma - Mass Spectrometry) were lead (Pb), arsenic (As), mercury (Hg), cadmium (Cd), chromium (Cr), nickel (Ni), molybdenum (Mo) and thallium (Tl), for a total of 151 measurements in 98 samples. The results showed that 11.3% of the measurements were quantifiable but below the legal maximum limits (MLs) set by EU regulations. The data obtained may be useful for dietary exposure information, inter-regional comparisons and for planning regional surveillance strategies.

Investigation of ochratoxin A in commercial cheeses and pork meat products by liquid chromatography-tandem mass spectrometry.

Ochratoxin A (OTA) is a mycotoxin produced by several species of Aspergillus and Penicillium and commonly detected in a wide range of foodstuffs. The purpose of this work was to monitor the presence of OTA in cheeses and pork meat products. A simple and accurate "dilute and shoot" method with no need of immunoaffinity column and isotopic labeled internal standard, by liquid chromatography-tandem mass spectrometry, was validated in accordance with the criteria set out in Commission Regulation (EC) No. 401/2006. The method showed good linearity in solvent and in matrix (R2  ≥ 0.995), limit of detection was 0.2 µg/kg for cheese and 0.3 µg/kg for pork meat products, limit of quantification was fixed at 1 µg/kg, and recovery was estimated at two different concentration levels (1 and 5 µg/kg) and ranged from 75% to 101%. The interday and intraday laboratory precisions were lower than 7%. The matrix effect, the recovery of the extraction process, and the overall process efficiency were evaluated. No significant ME was observed in the two matrices considered. This method was applied to the analysis of 75 samples, coming from official controls implemented by the Lazio Region (Central Italy). In one sample of dry-cured ham, the concentration found (69.3 µg/kg) was well above the guidance value recommended by the Italian Ministry of Health (1 µg/kg). These data together with the detection of OTA in three grated cheeses suggest the importance of monitoring these products. Considering the high dietary intake of these matrices, especially among vulnerable populations, further research should be devoted to estimate exposure and risk assessment for OTA.

Exploring the brain tissue proteome of TgCRND8 Alzheimer's Disease model mice under B vitamin deficient diet induced hyperhomocysteinemia by LC-MS top-down platform.

The multifactorial nature of Late Onset Alzheimer's Disease (LOAD), the AD form of major relevance on epidemiological and social aspects, has driven the original investigation by LC-MS and top-down proteomics approach of the protein repertoire of the brain tissue of TgCRND8 model mice fed with a diet deficient in B vitamins. The analysis of the acid-soluble fraction of brain tissue homogenates identified a list of proteins and peptides, proteoforms and PTMs. In order to disclose possible modulations, their relative quantification in wild type and AD model mice under both B vitamin deficient and control diets was performed. The levels of metallothionein III, guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-2 and brain acid soluble protein 1 showed statistically significant alterations depending on genotype, diet or both effects, respectively. Particularly, metallothionein III exhibited increased levels in TgCRND8 mice under B vitamin deficient diet with respect to wild type mice under both diets. Brain acid soluble protein 1 showed the opposite, revealing decreased levels in all diet groups of AD model mice with respect to wild type mice in control diet. Lower levels of brain acid soluble protein 1 were also observed in wild type mice under deficiency of B vitamins. These results, besides contributing to increase the knowledge of AD at molecular level, give new suggestions for deeply investigating metallothionein III and brain acid soluble protein 1 in AD.

High-resolution mass spectrometry for thymosins detection and characterization.

OBJECTIVES: The aim of this study was to characterize ß and α thymosins and their proteoforms in various tissues and bodily fluids by mass spectrometry and to look at their association with a wide variety of pathologies. METHODS: A top-down proteomic platform based on high-performance liquid chromatography (HPLC) coupled to high-resolution LTQ-Orbitrap mass spectrometry (MS) was applied to the characterization of naturally occurring peptides. RESULTS: In addition to thymosin ß4 (Tß4) and ß10 (Tß10), several post-translational modifications of both these peptides were identified not only in bodily fluids but also in normal and pathological tissues of different origins. The analysis of tissue specimens allowed the characterization of different C-terminal truncated forms of Tß4 and Tß10 together with other proteolytic fragments. The sulfoxide derivative of both Tß4 and Tß10 and the acetylated derivatives at lysine residues of Tß4 were also characterized. Different proteoforms of prothymosin α, parathymosin α, thymosin α1 and thymosin α11 together with diverse proteolytic fragments were identified too. CONCLUSION: The clinical and prognostic significance and the origin of these proteoforms have to be deeply investigated.

Integrated proteomic platforms for the comparative characterization of medulloblastoma and pilocytic astrocytoma pediatric brain tumors: a preliminary study.

A top-down/bottom-up integrated proteomic approach based on LC-MS and 2-DE analysis was applied for comparative characterization of medulloblastoma and pilocytic astrocytoma posterior cranial fossa pediatric brain tumor tissues. Although rare, primary brain tumors are the most frequent solid tumors in the pediatric age. Among them the medulloblastoma is the prevalent malignant tumor in childhood while pilocytic astrocytoma is the most common, rarely showing a malignant progression. Due to the limited availability of this kind of sample, the study was applied to pooled tumor tissues for a preliminary investigation. The results showed different proteomic profiles of the two tumors and evidenced interesting differential expression of several proteins and peptides. Top-down proteomics of acid-soluble fractions of brain tumor homogenates ascribed a potential biomarker role of malignancy to ß- and α-thymosins and their truncated proteoforms and to C-terminal truncated (des-GG) ubiquitin, resulting exclusively detected or over-expressed in the highly malignant medulloblastoma. The bottom-up proteomics of the acid-soluble fraction identified several proteins, some of them in common with 2-DE analysis of acid-insoluble pellets. Peroxiredoxin-1, peptidyl-prolyl cis-trans isomerase A, triosephosphate isomerase, pyruvate kinase PKM, tubulin beta and alpha chains, heat shock protein HSP-90-beta and different histones characterized the medulloblastoma while the Ig kappa chain C region, serotransferrin, tubulin beta 2A chain and vimentin the pilocytic astrocytoma. The two proteomic strategies, with their pros and cons, well complemented each other in characterizing the proteome of brain tumor tissues and in disclosing potential disease biomarkers to be validated in a future study on individual samples of both tumor histotypes.