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Publications (13) View all
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Article: Application of 2-D DIGE to formalin-fixed diseased tissue samples from hospital repositories: results from four case studies.
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ABSTRACT: PURPOSE: In the recent past, the potential suitability of fixed samples to 2-D DIGE studies has been demonstrated on model tissues, but not on "real-world" archival tissues. Therefore, this study was aimed to assess the quality of the results delivered by 2-D DIGE on samples retrieved from hospital tissue repositories. EXPERIMENTAL DESIGN: Diseased and normal tissue samples (namely, human gastric adenocarcinoma and normal gastric tissue, human lung neuroendocrine tumors, canine mammary tubulo-papillary carcinoma and normal mammary tissue, sheep liver with cloudy swelling degeneration and normal liver tissue) were retrieved from human and veterinary biorepositories and subjected to full-length protein extraction, cyanine labeling, 2-D DIGE separation, image analysis, MS analysis, and protein identification. RESULTS: Archival samples could be successfully subjected to 2-D DIGE, providing maps of satisfactory resolution, although with varying pattern complexity (possibly influenced by pre-analytical variables). Moreover, differentially expressed protein identities were consistent with the disease biology. CONCLUSIONS AND CLINICAL RELEVANCE: 2-D DIGE can support biomarker discovery and validation studies on large sample cohorts. In fact, although some information complexity is lost when compared to fresh-frozen tissues, their vast availability and the associated patient information can considerably boost studies suffering limited sample availability or involving long-distance exchange of samples.PROTEOMICS - CLINICAL APPLICATIONS 10/2012; · 1.81 Impact Factor -
Article: Evaluation of the suitability of archival Bouin-fixed paraffin-embedded tissue specimens to proteomic investigation.
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ABSTRACT: Bouin's solution has been used for over a century as a common fixative in several pathology laboratories worldwide. Therefore, a considerable number of Bouin-fixed paraffin-embedded (BFPE) tumor samples of various origin are available in hospital repositories as a powerful information mine for clinical investigations. To date, however, such archived tissues have not been subjected to a systematic study aimed to evaluate their potential use in proteomics. In this report, we investigated whether archival BFPE tissue specimens could be exploited for proteomic studies, upon application of protein extraction and proteomic analysis methods previously optimized for formalin-fixed samples. As a result, gastric BFPE protein extracts exhibited poor suitability for 2D-PAGE analysis, whereas over 300 unique proteins could be successfully detected when extracts were subjected to SDS-PAGE followed by LC-MS/MS (GeLC-MS/MS). Among these, several known markers for gastric cancer and normal gastric functionality were identified, indicative of biological and clinical significance of proteomic data mined from BFPE tissues. A quantitative and qualitative comparison of FFPE and BFPE tissue proteomes was also performed, and results are reported. In conclusion, we demonstrated that BFPE specimens can be analyzed by means of a proteomic approach such as GeLC-MS/MS. Although considerable molecular biases and technical constraints exist, BFPE tissue archives can be fruitfully exploited for gathering proteomic data from particularly precious samples.Electrophoresis 05/2012; 33(9-10):1375-84. · 3.30 Impact Factor -
Article: Comparison of blood serum peptide enrichment methods by Tricine SDS-PAGE and mass spectrometry.
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ABSTRACT: Characterisation of blood serum peptides can provide valuable information on physiological and pathological processes. However, the analysis of raw serum samples by MS results in the identification of a limited number of peptides. In order to improve sensitivity, many peptide enrichment methods have been proposed during the last ten years. Here, we present a comparison of fractionation methods aimed to simplify analysis of small proteins and peptides in blood serum, one of the most promising sources of putative biomarkers. Specifically, three methods based on ultrafiltration, differential precipitation, and peptide ligand libraries (ProteoMiner) were evaluated for the enrichment of peptides and low molecular weight proteins, as demonstrated by Tricine SDS-PAGE and subsequent LC-MS/MS (GeLC-MS/MS). As a result, differential solubilisation (DS) allowed the identification of the highest number of peptides. Moreover, the DS method enabled also the quantitative comparison of samples, producing fundamental information in biomarker discovery approaches.Journal of proteomics 07/2011; 75(1):93-9. · 5.07 Impact Factor -
Article: Proteomics and pathway analyses of the milk fat globule in sheep naturally infected by Mycoplasma agalactiae provide indications of the in vivo response of the mammary epithelium to bacterial infection.
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ABSTRACT: Milk fat globules (MFGs) are vesicles released in milk as fat droplets surrounded by the endoplasmic reticulum and apical cell membranes. During formation and apocrine secretion by lactocytes, various amounts of cytoplasmic crescents remain trapped within the released vesicle, making MFGs a natural sampling mechanism of the lactating cell contents. With the aim of investigating the events occurring in the mammary epithelium during bacterial infection, the MFG proteome was characterized by two-dimensional difference gel electrophoresis (2-D DIGE), SDS-PAGE followed by shotgun liquid chromatography-tandem mass spectrometry (GeLC-MS/MS), label-free quantification by the normalized spectral abundance factor (NSAF) approach, Western blotting, and pathway analysis, using sheep naturally infected by Mycoplasma agalactiae. A number of protein classes were found to increase in MFGs upon infection, including proteins involved in inflammation and host defense, cortical cytoskeleton proteins, heat shock proteins, and proteins related to oxidative stress. Conversely, a strikingly lower abundance was observed for proteins devoted to MFG metabolism and secretion. To our knowledge, this is the first report describing proteomic changes occurring in MFGs during sheep infectious mastitis. The results presented here offer new insights into the in vivo response of mammary epithelial cells to bacterial infection and open the way to the discovery of protein biomarkers for monitoring clinical and subclinical mastitis.Infection and immunity 06/2011; 79(9):3833-45. · 4.21 Impact Factor -
Article: Effects of postmortem storage temperature on sea bass (Dicentrarchus labrax) muscle protein degradation: analysis by 2-D DIGE and MS.
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ABSTRACT: Storage conditions are known to be important for postmortem deterioration of fish muscle, and temperature is one of the factors with the strongest impact on this process. In order to shed light on the influence of temperature on the status of sea bass (Dicentrarchus labrax) muscle proteins during postmortem storage, a 2-D DIGE and mass spectrometry study was performed on fish kept at either 1 or 18°C for 5 days. As expected, the greatest alterations in sea bass filet protein composition were observed upon postmortem storage at 18°C, with distinct changes appearing in the 2-D protein profile after 5 days of storage at this temperature. In particular, degradation of the myofibrillar protein myosin heavy chain and of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, among the most abundant muscle proteins, could be clearly observed upon storage at higher temperatures. Although to a lesser extent, however, several proteins were observed to vary in abundance also upon storage for 5 days at 1°C. In particular, one of the most interesting observations was the rapid and significant decrease in the abundance of nucleoside diphosphate kinase B and phosphoglycerate mutase 2, which was observed also at low storage temperatures and appeared to be temperature-independent. The results of this study offer new knowledge on changes occurring in sea bass muscle proteins during postmortem storage at different temperatures and provide indications on protein degradation trends that might be useful for monitoring freshness of fish and quality of storage conditions.Proteomics 05/2011; 11(14):2901-10. · 4.43 Impact Factor
