Proceedings and Report

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2 Proceedings and Report 76

3 Italian Proteomics Association 6 th Annual National Conference Rettorato dell Università degli Studi (via Verdi, 8) & Dipartimento di Biologia Animale e dell Uomo (via Accademia Albertina, 13) Turin, 21 st - 24 th June, 2011

4 ItPA Executive Board Scientific Committee Urbani Andrea (President) Bini Luca (Vice President) Roncada Paola (Treasurer) Modesti Alessandra (Secretary) Castagnola Massimo Fasano Mauro Organising Committee Pessione Enrica (President) Cavaletto Maria Fattori Paolo Giunta Carlo Nebbia Carlo Novelli Franco Timperio Anna Maria Pessione Enrica

5 The Organization and Scientific Committes acknowledge the support of: ABSciex Agilent Technologies Biorad Bruker Dionex GE Healthcare Thermo Scientific Waters

6 WORKSHOP PROGRAMME

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8 CONGRESS PROGRAMME

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11 LECTURES

12 L1 PROTEINS PRODUCED BY PROBIOTIC BACTERIA AS MEDIATORS OF HOST-BACTERIA INTERACTIONS. A FOCUS ON BIFIDOBACTERIA Abelardo Margolles IPLA - CSIC, Ctra. Infiesto s/n Villaviciosa, Asturias, Spain Bifidobacteria are commensal microorganisms of the human gastrointestinal tract which are largely being used in functional foods. Some strains are able to exert health-promoting effects, being considered as probiotics. The scarcity of genetic tools has hindered the development of functional genomic studies in bifidobacteria, like the identification of molecular mechanisms underlying their survival under different environmental challenges. However, some of these experimental obstacles have been successfully overcome with the use of proteomics. The aim of this presentation is to summarize some of the work carried out in our group during the last years, mainly focused on the study of the response of bifidobacteria to gastrointestinal conditions. Bile salts and acid ph represent a key challenge for bifidobacterial survival in the gastrointestinal environment and may, thus, determine their functionality at this location. However, the precise mechanisms employed by bifidobacteria to counteract their effects are poorly understood. The comprehension of these mechanisms may lead to better knowledge of host-microbiota interactions, and to a rational application of probiotic bifidobacteria. We have used proteomics and functional genomics techniques for a deeper understanding of the molecular mechanisms involved in bifidobacteria response to gastrointestinal stress. Acid response involves a number of changes jointly dedicated to mitigate the impact of protons in the cell physiology, mainly by controlling the intracellular ph through different mechanisms. On the other hand, exposure to bile in bifidobacteria produces deep changes in the carbohydrate metabolism, as well as changes in fatty acid biosynthesis and an increase in the amount of molecular chaperones and DNA protection proteins. Remarkably, bile also triggers the synthesis of several membrane proteins involved in the bile resistance phenotype of bifidobacteria. Finally, It is worth mentioning the role of some extracellular proteins from bifidobacteria able to act as intestinal adhesins. We have characterized a moonlighting protein which is an aggregation factor and a mucus binding protein, likely contributing to the colonization capacity of bifidobacteria in the human intestine. L2 PROTEOMICS DRIVEN TRANSLATIONAL RESEARCH IN PANCREATIC CANCER Francesco Novelli Centro Ricerche Medicina Sperimentale (CERMS) Azienda Universitaria Ospedaliera, San Giovanni Battista, Torino; Dipartimento Medicina e Oncologia Sperimentale, Università di Torino, Torino, Italy Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by rapid progression, invasiveness, and resistance to treatment. It is the fourth leading cause of cancer death with a 2% 5-years survival rate. This poor prognosis is due, in part, to a lack of early diagnosis, indeed biomarkers for the early detection of PDAC are lacking. By using a proteomic approach, defined as SERological Proteome Analysis (SERPA) we have defined a number of PDACassociated antigens that are specifically recognized by autoantibodies present in PDAC patients sera. These autoantibodies recognized with high frequency a number of metabolic enzymes and cytoskeletal proteins that are mostly up-regulated in PDAC. The protein recognized with the highest frequency by autoantibodies in PDAC sera was the glycolytic enzyme -enolase (ENOA). Accumulating evidences have revealed that ENOA is a multifunctional protein involved in many physiological and pathophysiological conditions. It is expressed on the surface of many prokaryotic and eukaryotic cells, where it acts as a plasminogen-binding receptor. This binding leads to activation of plasminogen to plasmin and it is critical in tumour cells spreading and tissue invasion. We have found that PDAC cell lines display ENOA on cell surface, where it could be critical in the invasion of PDAC cells through extracellular matrix. We found that treatment of PDAC cells with a mouse monoclonal antibody against human ENOA inhibited plasminogen-dependent migration both in vitro and in vivo. Microarray, Western blot and immunohistochemistry studies have indicated that ENOA is overexpressed in PDAC tissues. ENOA is able to elicit T cell proliferation, activation and cytotoxic specific T lymphocytes (CTL) differentiation both in vitro and in vivo. We have used genetically engineered mice (GEM) that spontaneously develop PDAC very similar to human to analyze the effect of DNA vaccination to human ENOA. Preliminary results indicated that DNA vaccination to ENOA elicits an integrated humoral and cellular immune response that significantly extends survival of GEM. Finally, six ENOA isoforms from pancreatic cell lines were identified by two-dimensional electrophoresis Western blot and mass spectrometry, and used as targets to screen the IgG reactivity of 120 PDAC patients, 40 healthy subjects, 50 non-pdac tumor patients, 46 chronic pancreatitis and 12 autoimmune diseases patients. Antibodies to two phosphorylated isoforms of ENOA (ENOA1,2) were observed in 62% of PDAC sera. Patients with antibodies to ENOA1,2 displayed better disease control upon therapy and longer survival. Our aim is to identify by mass spectrometry analysis the phosphorylated residues in ENOA1,2 isoforms and afterwards to find the epitopes of phosphorylated ENOA that are recognized by PDAC autoantibodies. This information will allow a short-term development of an assay based on ENOA phosphorylated peptides for rapid diagnosis of PDAC. As whole these data demonstrate that SERPA is a powerful strategy to identify tumor associated antigens, like ENOA, that are suitable for both therapeutic and diagnostic (theragnostic) purposes in PDAC.

13 L3 L4 AN OMICS APPROACH TO HUMAN LONGEVITY WITHIN A SYSTEMS BIOLOGY PERSPECTIVE Claudio Franceschi Dipartimento di Patologia Sperimentale dell Università di Bologna (Italy) PUSHING THE LIMITS OF PROTEOMICS TECHNIQUES TO UNDERSTAND STRESS AND TOLERANCE IN AN ALLOPOLYPLOID CROP Sebastien Carpentier K.U.Leuven / Research Foundation Flanders (FWO) Faculty of Bioscience Engineering Kasteelpark Arenberg 13 bus Leuven, Belgium Polyploidy and allopolyploidy have played an important role in the evolution of many plants and crops and it generates a great deal of protein polymorphism. Protein polymorphism is a driving force behind crop improvement and the analysis of protein isoforms is important in understanding crop biodiversity and stress tolerance. The combination of two-dimensional electrophoresis (2DE) and 2D DIGE with de novo MS/ MS sequence determination is still the workhorse for proteomics in nonmodel plants (Carpentier et al., 2008) and is an excellent tool to quantify and analyze the different protein isoforms and to identify variety specific proteins and amino acid polymorphisms (Carpentier et al., 2011). However, proteins with multiple membrane spanning domains a class of proteins extremely important in stress reactions- are hardly detected on classical 2-DE gels (Vertommen et al., 2010). Therefore we have also explored the possibility to apply a peptide based shot gun approach and pushed the limits (Vertommen et al., 2011). A peptide based approach in non-model plants demands special precautions to prevent false positive identification of proteins and the reconstruction of peptides into proteins is a big challenge. Therefore, we developed a workflow that included (i) optimization of protein extraction and the peptide separation (2DLC), (ii) the use of MSe and DDA for protein quantification and identification, (iii) performing de novo sequencing to allow a sequence homology search, (iv) visualization of identified peptide-protein associations using Cytoscape to remove redundancy and wrongly assigned peptides and to visualize the protein inference problem. References Carpentier, S., Panis, B., Vertommen, A., Swennen, R., Sergeant, K., Renaut, J., Laukens, K., Witters, E., Samyn, B., Devreese, B., Proteome analysis of non-model plants: a challenging but powerful approach. Mass Spectrometry Reviews, vol. 27, pp Carpentier, S. C., Panis, B., Renaut, J., Samyn, B., Vertommen, A., Vanhove, A.-C., Swennen, R., Sergeant, K., The use of 2Delectrophoresis and de novo sequencing to characterize inter- and intracultivar protein polymorphisms in an allopolyploid crop. Phytochemistry In Press. Vertommen, A., Panis, B., Swennen, R., Carpentier, S., Evaluation of chloroform/methanol extraction to facilitate the study of membrane proteins of non-model plants. Planta, vol. 231, pp Vertommen, A., Moller, A. L., Cordewener, J. H., Swennen, R., Panis, B., Finnie, C., America, A. H., Carpentier, S. C., A workflow for peptidebased proteomics in a poorly sequenced plant: A case study on the plasma membrane proteome of banana. J Proteomics In Press.

14 L5 L6 PLANT GENOMIC INFORMATIONAL CONTENT AT THE START OF THE POSTGENOMIC ERA Francesco Salamini Edmund Mach Fondation, Istituto Agrario di S. Michele all Adige e Parco Tecnologico Padano. After 10 years from the sequencing of the first plant genome, the current state of the field is here reviewed. Plant genomes can be relatively small but, nevertheless, they are the result of ancient and recent genome duplications (WGS); in addition, a common observation is that plant speciation frequently is the result of an allopolyploidy event. The genome status of these organisms is thus characterized by a large number of genes (for example, the recently sequenced apple genome contains more then 57,000 genes, a number deriving mainly from a 50 million years old duplication of almost all chromosomes). The presentation first introduces the major conclusions learned from the sequencing of the apple and grape genomes, as carried out at the Mach Foundation, Istututo Agrario di S. Michele all Adige, Trento. Of relevant interest in these two species are genes controlling disease resistance (365 in grape and 992 in apple) and those responsible for product quality (62 and 110 genes encoding enzymes responsible for fenolics and terpenoids in grape; 1,246 genes related to volatiles, aromatic-compounds, pigments, antioxidant and sorbitol biosynthesis in apple). A summary of relevant results deriving from the two mentioned species, compared to those of other 17 plant genomes today available, is also introduced. Of relevance to the proteomic meeting are two particular issues: the amplification in plants of secondary metabolism-related genes, which supports a large redundancy of some gene families (this creates problems when associating specific genes to the proteins they encode); the second issue concerns RNA and DNA sequence differences at the exonic level, a finding which call for more subtle and effective protein identification methods. nextprot, THE HUMAN PROTEIN KNOWLEDGE PLATFROM IN THE CONTEXT OF THE HUMAN PROTEOME PROJECT. Amos Bairoch Swiss Institute of Bioinformatics and University of Geneva In September 2008, the UniProt/Swiss-Prot group achieved a major milestone: the first complete manual annotation of the full set of human proteins (derived from about 20'000 genes). This corpus of data which is quite rich in information pertinent to modern biomolecular medical research, clearly shows that there is a large gap in our knowledge of human proteins in terms of functional information as well as protein characterization (PTMs, protein/protein interactions, subcellular locations, etc). This gap resides not only in the available experimental information, but also in the way this information has been stored, which is far from being sufficient to help researchers making sense of what all these human proteins do in our bodies! Therefore, in the framework of CALIPHO, a new interdisciplinary group created jointly by the University of Geneva and the SIB, we are developing nextprot, a new humancentric protein knowledge resource, with the aim to help researchers answer pertinent questions. To achieve this goal we need to build up on a corpus of both curated knowledge -originating mainly from the UniProtKB/ Swiss- Prot knowledgebase -and carefully selected and filtered highthroughput data pertinent to human proteins. Such a data gathering and grading effort are complemented by the development of tools that allow such data to be analyzed. nextprot is a common development of the SIB and of GeneBio SA. In January 2011 we came out with the first release of nextprot (available at beta.nextprot.org). We will present its current content and what we are planning to add in the near future. This is especially important in the context of HPP as we plan to develop nextprot as one of the knowledge pillar of this crucial scientific initiative. We hope you will enjoy working with nextprot and will help us making it evolve by telling us of your specific needs.

15 ABSTRACTS

16 1.O.1 1.O.2 FARM ANIMAL PROTEOMICS: A COST ACTION Paola Roncada* Istituto Sperimentale Italiano Lazzaro Spallanzani, Milano The use of proteomic strategies to investigate animal health and disease has been limited by the lack of international coordination and collaboration. The COST action FAP- Farm Animal Proteomics, FA1002, that is started in November 2010, form a network of the leading European scientists who are focused on farm animal proteomics, but it is also designed to encourage participation of researchers who are at an early stage of utilizing the range of proteomic methods which are becoming available. This network benefit the European economy by providing advanced analytical tools to enhance animal production, health and welfare, as well as in the assessment of food quality and safety related to the protein in food produced from animal origin. The action works through three major workgroups: wg1 proteomics and animal health, that focus on research relevant to animal health covering biomarkers of infectious, parasitic and metabolic diseases,. The WG2, proteomics of food of animal origin- provide communication in fundamental research in proteomics related to food production, quality and food safety. The focus of Working Group 3, Advancing Methodology for Farm Animal Proteomics - is about technical aspects of animal proteomics, from sample collection/ preparation to protein separation, identification, and quantification in production animal body fluids, tissues and post harvest derived products. The action organizes meeting every year and the dissemination of results of the collaboration are published in the website that is the main source of informations. Challenge of the action is also to use EU found to help young scientists to join several laboratory in different EU COST member. ANALYSIS OF THE SECRETOME OF THE GROUP B STREPTOCOCCUS Roberta Galbo a, S. Papasergi b, M. Garibaldi b, I. Pernice a, C. Lo Passo a, F. Mandanici b, L. Romeo b, P. Trieu-Cuot c, G. Teti b and C. Beninati b a Department of Life Sciences, University of Messina, Italy b The Elie Metchnikoff Department, University of Messina, Italy c Unit of Gram Positive Pathogens Biology, Pasteur Institute, Paris, France Group B streptococci (GBS), are important human pathogens, particularly affecting neonates. In recent years, proteomics analysis of surface proteins of group A and B streptococci has greatly contributed to the identification of novel virulence factor and candidate vaccines. In the present study we examined for the first time, by proteomics, the complex of the covr/covs (covr/s) two component system, since inactivation of this system is thought to mimic the physiological status of the GBS infections phase. We identified a total of 37 proteins, about 95% of which were predicted to be secreted or surface-associated by informatics analysis. Fourteen of the 37 identified proteins were secreted by both strains, while 13 were found exclusively in DcovRS supernatants. The latter comprised, by informatics analysis, previously unstudied cell wall associated proteins and/or homologs of putative virulence factors 1,2,3,4 present in other bacterial species. Our data suggest that proteomics analysis of the GBS secretome might be a useful tool for the rapid identification of novel virulence factors. Corresponding author: Paola Roncada, PhD Istituto Sperimentale Italiano Lazzaro Spallanzani Sezione di Proteomica c/o Facoltà di Medicina Veterinaria Via Celoria 10 Milano Italy [email protected] References: Roberta Galbo Researcher and Aggregate professor of Genetic Dept. of Life Science, University of Messina, Italy Salita Stagno D Alcontres 31, Messina, Italy [email protected] Tel Fax References: [1] Tiwari R, Qin A, Artiushin S, Timoney JF, Se 18.9, an anti-phagocytic factor H binding protein of S. Equi, Veterinary Microbiology, 2006, 121 (1-2): [2] Zhu H, Liu M, Sumby P, Lei B, The secreted esterase of Group A streptococcus is important for invasive skin infection and dissemination, Infection and Immunity, 77(12): [3] Liu M, Zhu H, Zhang J, Lei B, Active and passive immunizations with the streptococcal esterase Sse protect mice against subcutaneous infection with GAS, Infection and Immunity, 2007, 75(7): [4] Buchanan JT, Simpson AJ, Aziz RK, Liu GY, Kristian SA, Kotb M, Feramisco J, Nizet V, DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps, Current Biology, 2006, 16(4):

17 1.O.3 SOIL PROTEOMICS: INTERFERENCE OF SOIL SOLID PHASES ASSESSED BY MODEL STUDIES Laura Giagnoni 1, Andrea Migliaccio 1, Loretta Landi 1, Daniel van der Lelie 2, Safyh Taghavi 2, Paolo Nannipieri 1, Giancarlo Renella 1 1 Department of Plant, Soil and Environmental Sciences, University of Florence, Florence, Italy 2 RTI International, Center for Agricultural and Environmental Biotechnology, 3040 Cornwallis Road Research, Triangle Park, NC 27709, USA Soil proteomics is a very promising approach for a deep understanding of the ecological and biological functions expressed by the highly diverse soil microbial communities, responsible for the soil functionality. Previous soil studies demonstrate that a critical point for implementing soil proteomics is the possibility to determine the location of the proteins extracted from soil, as most of the soil constituents (e.g. sand, clay hydrooxides, humic substances) can stabilize proteins actively or passively released by the soil microorganisms, making difficult to distinguish between newly expressed and soil-stabilized proteins. To better understand the potentials of soil proteomics and specifically address the effects of various soil solid phases on direct protein extraction from soil, we have used a model study, based on the use of the Gram-negative soil-borne bacterium Cupriavidus metallidurans CH34, with known genome and proteome, inoculated into microcosms containing quartz sand, clays, humic acids and an artificial soil. The bacterial proteome was analyzed by 2-DE and mass spectrometry approach. Based on the previous results which showed that the presence of the high reactive clay montmorillonite gave the largest interference in the bacterial proteome analysis as compared to low reactive clay kaolinite, we also inoculated C. metallidurans CH34 in artificial soils with a different kaolinite:montmorillonite ratios. The results from this study showed that the presence of high reactive clays in soils may negatively influence the potential of recognize the proteomic signature of bacteria in soil microbial communities, likely due to irreversible proteins adsorption. We conclude that more model studies are needed to improve the protein extraction and purification from soils. Corresponding author: Dr. Laura Giagnoni, Department of Plant, Soil and Environmental Sciences, Piazzale delle Cascine, Firenze (FI), Italy, [email protected]; tel O.1 IN VITRO VITAMIN K TREATMENT IS NOT CAPABLE TO RESQUE UNEFFICIENT MGP-CARBOXYLATION IN PXE FIBROBLASTS Giulia Annovi, Federica Boraldi, Daniela Quaglino Dept. Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy Abnormal soft connective tissue mineralization has been related to mutations in the MGP (Matrix Gla Protein) gene or as a consequence of an insufficient vitamin K-dependent gamma-carboxylation of specific Glaresidues on the MGP protein. Interestingly, low levels of vitamin K and reduction of carboxylated-matrix Gla Protein (Gla-MGP) have been demonstrated in the plasma of patients affected by pseudoxanthoma elasticum (PXE), a rare genetic disorder associated to mutations in the ABCC6 gene, and characterized by the progressive mineralization of elastic fibers. Consistently, we have recently reported that fibroblasts, isolated from PXE patients and cultured in vitro, have an altered protein profile exhibiting an insufficient vitamin-k-dependent carboxylation of MGP, a down-regulation of protein disulfide isomerase (PDI) and an upregulation of calumenin (CALU), two proteins of the endoplasmic reticulum that are involved in vitamin K cycle, by assuring the appropriate reduced environment, necessary for regenerating the vitamin in its active form, and by inhibiting the gamma-carboxylase activity, respectively. It has been therefore suggested that vitamin K supplementation might be capable to restore MGP carboxylation in PXE and to inhibit and/or delay the pathologic occurrence of the calcification process. However, very recent studies in the PXE animal model (abcc6-/- mice) failed to demonstrate the efficacy of vitamin K supplementation in counteracting the mineralization of soft connective tissues, thus rising several questions on the ability of cells to utilize the vitamin and to efficiently activate MGP restoring the appropriate environment. Aim of the present study was to investigate, in vitro, the effects of vitamin K1 and K2 supplementation on control as well as on PXE fibroblasts, used as a useful in vitro model to monitor the ability of the vitamin to counteract abnormal MGP carboxylation. Data from the present study indicate that both control and PXE fibroblasts are able to utilize vitamin K1 and K2 and to significantly increase protein carboxylation, however neither vitamin K1 or vitamin K2 supplementations are capable to inhibit the mineralization process and to positively modulate MGP, its expression being down-regulated at high doses of vitamin K2. It can be suggested that the role of vitamin K1 and especially of vitamin K2 on the calcification process is mediated by changes in the carboxylation as well as in the expression of MGP, and that other factors, possibly involving phosphate deposition, should be taken into consideration for an effective inhibition of the mineralization process in peripheral tissues. Prof. Daniela Quaglino Dipartimento Scienze Biomediche Via Campi 287, Modena, Italy [email protected] Te /5446

18 2.O.2 SEQUENTIAL APPLICATION OF URINARY PEPTIDE CLUSTERS OBTAINED AFTER WEAK-CATION EXCHANGE MAGNETIC BEAD PURIFICATION TO DETECT RENAL CELL CARCINOMA Erica Gianazza 1,2, Yuri E.M. van der Burgt 2, Italo Zoppis 3, Massimiliano Borsani 3, Giancarlo Mauri 3, Clizia Chinello 1, Valeria Squeo 1, Giancarlo Albo 4, Stefano Signorini 5, André M. Deelder 2, Marzia Galli Kienle 1 and Fulvio Magni 1,* 1 Dept. of Experimental Medicine, University of Milano- Bicocca, Monza, Italy. 2 Dept. of Parasitology, Biomolecular Mass Spectrometry Unit, LUMC, Leiden, The Netherlands. 3 Dept. of Informatics, Systems and Communication, University of Milano-Bicocca, Milano, Italy. 4 Dept. of Specialistic Surgical Sciences, Urology Unit, University of Milano, Ospedale Maggiore Policlinico Foundation, Milano, Italy. 5 Desio Hospital, University of Milano-Bicocca, Milano, Italy. Renal Cell Carcinoma (RCC) is the most common kidney malignancy. Since RCC is highly resistant to radio- and chemotherapies patients have a poor prognosis. Therefore, biomarkers for early diagnosis are urgently needed. Previously we have identified various peptides in serum and urine of RCC patients that discriminate from healthy individuals with very high diagnostic potential. In this study we have extended the investigation to patients with a non-rcc tumor using a combination of weak cation exchange magnetic bead (WCX-Mb) purification of urine and matrixassisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). Urinary protein profiles of 85 controls (Ctrl), 79 clear cell RCC (RCC) and 23 non-rcc patients (non-rcc) were obtained and statistically evaluated aiming for detection of potential biomarkers. Multiple peptides were observed with intensity / area differences large enough to distinguish controls from patients. Two clusters were build based on a Support Vector Machine (SVM) algorithm containing three and five m/z-values, respectively. The SVM clusters allow the discrimination between the three classes through a sequential decision process. We achieved a significant rate of predicted values (i.e., precision ratios greater than 80%) with good specificity and sensitivity (i.e., recall ratios greater than 77%) for both clusters by considering their discrimination task. Moreover the application of a Genetic Algorithm (GA) resulted in similar classification performance. In conclusion, the results indicate that peptides extracted using WCX display a diagnostic capability to discriminate RCC patients from controls. Moreover the m/z-values included in the clusters may be used to detect non-rcc tumors. The present work has been supported by grant FIRB n. RBRN07BMCT of the Italian Ministry of Research and by grant IIT Project SEED: IPG- CHIP. Prof. Fulvio Magni Dept. of Experimental Medicine, University of Milano-Bicocca Via Cadore 48, Monza, Italy [email protected] Phone: Fax: O.3 MODULATION OF URINARY PEPTIDOME IN HUMANS EXPOSED TO HIGH ALTITUDE HYPOXIA Veronica Mainini A*, Erica Gianazza A*, Clizia Chinello A, Grzegorz Bilo B, Miriam Revera B, Andrea Giuliano B, Gianluca Caldara B, Carolina Lombardi B, Alberto Piperno C, Fulvio Magni A, Gianfranco Parati B, on behalf of HIGHCARE investigators A Department of Experimental Medicine, University of Milano-Bicocca, Monza, Italy B Department of Cardiology, S. Luca Hospital, IRCCS, Istituto Auxologico Italiano, Milan, Italy C Department of Clinical Medicine and Prevention, University of Milano-Bicocca, Monza, Italy *equally contributing author. The exposure of healthy subjects to high altitude represents a good model to explore the pathophysiology of diseases related to tissue hypoxia and is a good model to evaluate pharmacological approaches potentially useful as a therapy for chronic diseases related to hypoxia. The aim of our study was to explore the urinary peptidome to detect alterations induced by the exposure of subjects to different altitudes (sea level, 3500 and 5400 mt a.s.l.) and/or to pharmacological treatment. Urines were collected from 47 subjects, randomly and blindly assigned to Placebo (n=24) or Telmisartan treatment (n =23). Samples were purified by the use of hydrophobic interaction magnetic beads, then analysed by MALDI-TOF MS. Results showed that the urinary peptidome is not affected by the administration of Telmisartan, neither at sea level nor at altitude. Conversely, 6 peptides were demonstrated to be differentially expressed in association with the exposure to altitude. In detail, we assessed that two of these peptides were down regulated at high altitude, suggesting molecules involved in the early phase of acclimatization. Other two peptides, instead, were demonstrated to be up-regulated at 5400 mt, suggesting a later modulation process. Finally, two peptides were downregulated at 3500 mt compared to sea level, but were comparable at sea level and at 5400 mt. These results suggest a potential late and compensatory adaptation to hypobaric hypoxia. This is the first study describing modulation of the urinary peptidome in response to the exposure to hypobaric hypoxia and could provide useful information on acclimatization processes. The present work has been supported by grant FIRB n. RBRN07BMCT of the Italian Ministry of Research. Veronica Mainini, PhD Biomedical Technologies Department of Experimental Medicine, University of Milano-Bicocca, Via Cadore 48, Monza (MB), Italy address: [email protected] Phone: Fax:

19 3.O.1 3.O.2 EFFECTS OF AGEING IN MUSCLE TISSUE: THE CONTRIBUTION OF PROTEOMICS Daniele Capitanio1, Michele Vasso1,2, Cecilia Gelfi1,2 1 Department of Sciences and Biomedical Technologies, University of Milan, Segrate (MI), Italy. 2 Institute of Bioimaging and Molecular Physiology, National Research Council, Segrate (MI), Italy. Ageing induces a progressive functional decline affecting the entire organism, therefore, the loss of muscle mass and function (sarcopenia) contributes significantly to a loss of functional autonomy. In muscles, the age-related degenerative changes produce alteration of morphology associated to muscle fiber atrophy, loss of satellite cells, and remodelling of neuronal structures. Morphological changes induced by ageing indicate a decrement of muscle fiber size, a change in fiber type distribution and appearance of mitochondrial aggregates with an increment of lipofuscin. This functional and morphological changes are also observed in nerves, in which ageing induces biochemical, morphological and functional variations both in myelin and in axons. Beside that, mitochondrial metabolism plays a pivotal role in aging, as its alteration influences the muscle function. In particular, the two morphologically distinct mitochondrial sub-fractions, named sub-sarcolemmal (SS) and intermyofibrillar (IMF) according to their diverse cellular localization, play different roles in muscle tissue. The modulation of the respiration rate, proteins and lipids composition, and the diverse biochemical (e.g. protein import) properties, contributes to muscle cell adaptation during ageing. The SS sub-fraction is more influenced by muscle changes and is more affected by ageing. Proteomics has been utilized to monitor, at the molecular level, the decline of muscle tissue, nerves innervating it and mitochondrial machinery Corresponding author: Cecilia Gelfi Associated Professor Università degli Studi di Milano Via F.lli Cervi, Segrate (MI) [email protected] Tel Fax PROTEOMIC INVESTIGATIONS ON DIFFERENTIAL POST-TRANSLATIONAL MODIFICATIONS OF TRANSTHYRETIN IN MULTIPLE SCLEROSIS Damiana Pieragostino a,b, Piero Del Boccio a,b, Maria Di Ioia c, Sonia Bucci a,b, Luisa Pieroni d,e, Simona D Aguanno d,e, Viviana Greco d,e, Diego Centonze d, Giovanna De Luca a,c, Carmine Di Ilio a,b, Alessandra Lugaresi a,c, Paolo Sacchetta a,b, Andrea Urbani a,d,e acentro Studi sull Invecchiamento (Ce.S.I.), Fondazione Università G. d Annunzio, Chieti, Italy bdip. di Scienze Biomediche, Università G. d Annunzio di Chieti e Pescara, Italy cdip. di Neuroscienze ed Imaging, Università G. d Annunzio di Chieti e Pescara, Italy dcentro Europeo Ricerca sul Cervello (CERC), IRCCS- Fondazione S. Lucia, Roma edip. di Medicina Interna, Università di Roma Tor Vergata, Roma, Italy Multiple Sclerosis (MS) is a multifactorial disease involving central nervous system that result in global health issues. Thus, the need of understanding the molecular machinery involved in this pathology to develop more effective pharmacological therapeutics. Cerebrospinal Fluid (CSF) is a helpful biological fluid to study MS as it collects molecules released from various cell types as a result of the pathology. By studying CSF of MS patients through proteomic approaches we described two non canonical oxidative post-traslactional modifications on CSF Transthyretin (TTR), a 55 kda omotetrameric protein involved in Thyroid Hormone (TH) transport into central nervous system (up to 80% in man). These isoforms, reconductable to Thio-cysteine and Sulpho-cysteine adducts, discriminate MS patients vs subjects with other neurological diseases. These non-canonical isoforms were not highlighted in serum of MS patients, showing a specific phenomenon of CNS. Western Blot analysis of TTR, in non-denaturing conditions, showed a correlation between the modified TTR and an atypical TTR dimer in CSF samples. These results could be very interesting for to better understand the role of TTR in TH transport in MS disease, since TH physiologically helps the oligodendrocytes precursor to turn into re-myelinating oligodendrocytes regulating fundamental cell activities, including cell cycle. Hence TTR redox imbalance might eventually modify TTR transport capacity in multiple sclerosis. This work is supported by FISM Research Fellowship Cod.2010/B/14. Damiana Pieragostino, Ph.D. Department of Biomedical Sciences "G. d'annunzio" University Via dei Vestini 31, Chieti Pescara, Italy [email protected] phone: Fax: Mobile:

20 3.O.3 4.O.1 PROTEOMIC PROFILING OF BRAIN STEROID 5 ALPHA REDUCTASES IN SLEEP-DEPRIVATED RATS. Alessio Soggiu*1, Marco Bortolato2, Paola Devoto1, Cristian Piras3, Luigi Bonizzi4, Paola Roncada5 1 Section of Neurophysiology and Neurochemistry, Department of Neurosciences B.B. Brodie, University of Cagliari, (Cagliari), Italy 2 Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles 3 Department of Zootechnical Science, University of Sassari, Italy 4 DIPAV, Facoltà di Medicina Veterinaria, Università degli Studi di Milano 5 Istituto Sperimentale L. Spallanzani, Milano, Italy. Steroid 5α reductase (S5αR) is rate-limiting enzyme of one of two major metabolic pathways in brain steroidogenesis. Recent evidence indicates that neuroactive steroids may involved in pathogenesis of schizophrenia spectrum disorders. Moreover, S5αR inhibition has been shown to induce therapeutic effects in animal models of schizophrenia and in several disorders associated to dopaminergic hyperreactivity (1). In rodents, sleep deprivation (SD) is known to induce a series of behavioural patterns, including sensory-motor gating deficit, which might be reflective of psychosis and mania and are countered by antipsychotics (2). The aim of this study was to evaluate, the impact of SD on expression levels of S5αR isozymes in rat brain. After 72 h of SD, rats were sacrificed and brain areas dissected out. Quantitative 1D and 2D western blotting (WB) with antibodies were performed on four brain areas of both control group and SD group (n = 8 each): medial prefrontal cortex (mpfc), nucleus accumbens (nacc), ippocampus (IPPO) and amigdala (AMG). 1D and 2D WB revealed that the expression of S5αR-1 and S5αR-2 was significantly augmented in SD animals in comparison to controls (p < 0.05) in mpfc and nacc area. In IPPO and AMG expression of S5αR-1 and S5αR-2 was unchanged in SD animals in comparison to controls. Regarding therapeutic effect of S5αR inhibition on gating deficit, data suggest that S5αR increase might cause altered balancing in neurosteroid levels, and it could be responsible of behavioral disruption observed in SD animals. SIALYLATION DEFINING CELL INVASIVENESS IN BREAST CANCER Giuseppe Palmisano1, Rikke Leth-Larsen2, Martin Larsen1 1Department of Biochemistry and Molecular Biology, The University of Southern Denmark, Odense, Denmark; 2Department of Cancer and Inflammation Research, Institute of Molecular Medicine, University of Southern Denmark Background: breast cancer is the leading female cancer in Europe and USA with the metastasis as the main reason for mortality. Aberrant sialylation on the cell surface has been correlated with poor prognosis and metastatic spread of several human malignancies. Due to that, understanding the sialobiology of tumor cells (oncosialobiology) with the identification of tumor-specific sialylated glycoprotein alterations will add important knowledge to breast tumor therapy. Methods: human breast tumour cells were transplanted into mice and cell line was selected for enhanced growth rate, faster metastasis using GFP probe and animal survival rate. The selected isogenic non-metastatic (NM2C5) and metastatic (M4A4) cell lines were grown in SILAC medium. After cell lysis and membrane protein extraction, hydrazide chemistry and a new method based on the combination of TiO2-HILIC chromatography were applied to enrich and quantitatively map the N-linked glycoproteome using MS-based detection. The quantitative glycan analysis was performed using an array of SA-specific lectins and GPCµLC-MS/MS in negative ion mode, using a LTQ-Orbitrap mass analyser. Results: Oncosialobiology is a new point of view of investigating the link between sialylation and metastasis using MS-based techniques and the SA-containing oligosaccharides represent a molecular target for cancer therapy. Moreover we have investigated the potential pitfalls in large scale N-linked glycoproteomic analyses providing new solution to avoid them. *Corresponding author. Giuseppe Palmisano; Institution: Department of Biochemistry and Molecular biology, University of Southern Denmark. Address: Campusvej 55, DK-5230, Odense M, Denmark. [email protected] Ph: *Corresponding author Alessio Soggiu, PhD Department of Neurosciences B.B. Brodie, Section of Neurophysiology and Neurochemistry University of Cagliari, Monserrato (CA) [email protected] Phone: Fax: References 1) Paba S, Frau R, Godar SC, Devoto P, Marrosu F, Bortolato M (2011) Curr Pharm Des. 17:151. 2) Frau R, Orrù M, Puligheddu M, Gessa GL, Mereu G, Marrosu F, Bortolato M (2008) Int J Neuropsychopharmacol. 11:947. Acknowledgements This work was supported by a fellowship financed by RAS (Regione Autonoma della Sardegna, PO Sardegna FSE L.R. 7/2007 Promozione della ricerca scientifica e dell innovazione tecnologica in Sardegna )

21 4.O.2 PEPTIDOME PROFILING OF INDUCED SPUTUM BY MESOPOROUS SILICA BEADS AND MALDI-TOF MS FOR BIOMARKER DISCOVERY OF INFLAMMATORY LUNG DISEASES Rosa Terracciano a, Mariaimmacolata Preianò a, Grazia P. Palladino b, Giovanna E. Carpagnano b, Maria P. Foschino Barbaro b, Girolamo Pelaia a, Rosario Maselli a and Rocco Savino a a Department of Experimental and Clinical Medicine, Laboratory of Mass Spectrometry and Proteomics, University of Catanzaro Magna Græcia, Catanzaro, Italy b Institute of Respiratory Disease, University of Foggia, Foggia, Italy Induced sputum is recognized as being of increasing importance for the diagnosis and monitoring of chronic inflammatory lung diseases. The main purpose of this study is to provide a valid approach to better fractionate and characterize the still under-estimated low-molecular weight proteome of induced sputum by using mesoporous silica beads (MSB) SPE coupled to MALDI-TOF MS. Sputum peptides were captured from both derivatized and non-derivatized MSB and then profiled by MALDI-TOF MS. Depending on the chemical groups present on the mesoporous surface, complex peptide mixtures were extracted from induced sputum and converted into reproducible MALDI profiles. The number of peaks detected as a function of S/N was evaluated for each mesoporous surface. More than 400 peaks with a S/N> 5 were obtained in comparison to 200 peaks detected without MSB. Additionally, as a proof-of-principle, we investigated the ability of this platform to discriminate between the sputome of patients with asthma and chronic obstructive pulmonary disease (COPD), and between these groups and those of healthy control subjects. Six m/z peaks emerged as potential diagnostic peptidic patterns able to differentiate these inflammatory airway diseases in the sputome range. Human alpha-defensins (HNP1, HNP2, HPN3) and three C-terminal amidated peptides, one of which is phosphorylated on serine, were identified by MALDI-TOF/TOF MS. These findings may contribute to defining a of an high-throughput screening MSbased platform for monitoring key peptidic-biomarkers for inflammatory and chronic respiratory diseases in induced sputum samples. Rocco Savino, Full Professor of Molecular Biology, Department of Experimental and Clinical Medicine, Laboratory of Mass Spectrometry and Proteomics, University Magna Græcia, University Campus, Europa Avenue, 88100, Catanzaro, Italy [email protected] phone fax O.3 GENOCOP ALGORITHM AND HIERARCHICAL GRID TRANSFORMATION FOR IMAGE WARPING OF TWO DIMENSIONAL GEL ELETROPHORETIC MAPS Emilio Marengo a, Elisa Robotti a, Marco Demartini a, Marina Cocchi b, Daniela Cecconi c a University of Eastern Piedmont, Department of Environmental and Life Sciences, Alessandria, Italy b University of Modena and Reggio Emilia, Department of Chemistry, Modena, Italy c University of Verona, Dept. of Biotechnology, Mass Spectrometry and Proteomics Lab, Verona, Italy As commonly acknowledged, two dimensional gel-electrophoresis is a multistep technique that requires an interdisciplinary approach to provide a final reliable result, expressed in terms of a series of candidate biomarkers to be further investigated via mass-spectrometry. This presentation focuses on the computational aspects of the analysis of 2D- PAGE images: the algorithms that are usually exploited to accomplish image warping and matching play in facts a fundamental role on the final result. It is therefore important to adopt reliable and robust algorithms that avoid matching mistakes that reflect on the final spot volume dataset. Here a method is proposed and applied to 2D-image warping. Hierarchical Grid Transformation is a powerful approach to SDS 2DPAGE maps warping. The hierarchy of the warping transformation is able to model both global and local deformations of the gels and the algorithm can be stopped when a desired degree of accuracy in the image alignment is obtained. The numerical optimization of the position of the nodes of the grid that are responsible of the image warping is a multivariate task that can be solved efficiently using Genetic Algorithm. The use of Genetic Algorithm ensures that an optimal position of the nodes can be defined with a low computational cost with respect to other methods. The optimal positions of the nodes of the grid can be successfully used for defining a good warping of the gels. Elisa Robotti, PhD University of Eastern Piedmont, Department of Environmental and Life Sciences Viale Michel 11, Alessandria, Italy [email protected] Tel: Fax: References [1] Daszykowski M, Stanimirova I, Bodzon-Kulakowska A, Silberring J, Lubec G. Start-to-end processing of two-dimensional gel electrophoretic images. J. Chromatogr. A. 2007; 1158: [2] Salmi J, Aittokallio T, Westerholm J, Griese M, Rosengren A, Nyman TA, Lahesmaa R, Nevalainen O. Hierarchical grid transformation for image warping in the analysis of two dimensional gel electrophoresis gels, Proteomics 2002; 2: [3] Davis L. Handbook of Genetic Algorithms. New York: Van Nostrand Reinhold; [4] Daszykowski M, Færgestad EM, Grove H, Martens H, Walczak B. Matching 2D gel electrophoresis images with Matlab Image Processing Toolbox. Chemom. Intell. Lab. Syst. 2009; 96: [5] Cecconi D, Zamò A, Parisi A, Bianchi E, Parolini C, Timperio AM, Zolla L, Chilosi M. Induction of Apoptosis in Jeko-1 Mantle Cell Lymphoma Cell Line by Resveratrol: A Proteomic Analysis. J. Prot. Res. 2008; 7: [6] Marengo E, Robotti E, Cecconi D, Hamdan M, Scarpa A, Righetti PG. Identification of the regulatory proteins in human pancreatic cancers treated with Trichostatin-A by 2D-PAGE maps and Multivariate Statistical Analysis. Anal. Bioanal. Chemistry 2004; 379: [7] Gustafsson JS, Blomberg A, Rudemo1 M. Warping two-dimensional electrophoresis gel images to correct for geometric distortions of the spot pattern. Electrophoresis 2002; 23:

22 5.O.1 ARSENIC STRESS IN PTERIS VITTATA ROOTS COLONIZED BY ARBUSCULAR MYCORRHIZAL FUNGI Elisa Bona, Chiara Cattaneo, Patrizia Cesaro, Francesco Marsano, Graziella Berta and Maria Cavaletto DISAV Dept Scienze dell Ambiente e della Vita Università del Piemonte Orientale A. Avogadro Alessandria Italy Pteris vittata is an arsenic hyperaccumulating plant and can tolerate very high arsenic concentration in soil. These qualities make P. vittata an ideal plant for phytoremediation purposes. Fern roots are the unique access point for the metalloid, so it is extremely important to investigate the effects on root proteome in presence of arsenic contamination. Moreover our approach has been focused to evaluate if arbuscular mycorrhizal (AM) symbiosis could modify protein expression profile. AM symbiosis is a widespread mutualism in nature, AM fungi colonize plant roots, improving plant mineral nutrition and promoting plant tolerance/resistance to biotic and abiotic stresses. We have studied the effects of the AM fungus Funneliformis mosseae (ex Glomus mosseae) on arsenic-treated P. vittata plants by proteomic tools. AM symbiosis induced a significant decrease of arsenic content in roots. Arsenic treatment affected the expression of 14 spots, the combination of arsenic and F. mosseae modulated 3 spots, while F. mosseae alone modulated 17 spots. Comparative analysis of arsenic- and mycorrhizalinduced proteins in P. vittata roots revealed that glycolytic enzymes were strongly involved. Moreover up regulation of aldehyde dehydrogenase represented a response to oxidative stress. In presence of F. mosseae colonization and arsenic, a decrease of S-adenosylmethionine synthase was detected, suggesting an alleviating effect for mycorrhization (1). To conclude, stress symptoms were observed after arsenic treatment even in the arsenic-hyperaccumulator P. vittata, while AM symbiosis sorted a protective effect toward arsenic stress. Corresponding author: Bona E. PhD V.le T. Michel 11, Alessandria, Italy. [email protected] Phone Fax Bona E. et al. (2011) Journal of Proteomics doi: /j.prot O.2 PROTEOMIC ANALYSIS OF A SPRING WHEAT CULTIVAR IN RESPONSE TO PROLONGED COLD STRESS Sara Rinalducci a, Maria Giulia Egidi a, Ghasem Karimzadeh b, Ferdous Rastgar Jazii c, Lello Zolla a adepartment of Environmental Sciences, University of Tuscia, Viterbo, Italy bplant Breeding and Biotechnology Department, Tarbiat Modares University, Tehran, Iran cnational Institute of Genetic Engineering and Biotechnology of Iran (NIGEB), Tehran, Iran Cold represents one of the major abiotic factors influencing plant growth and development worldwide. We analysed the long-term responsiveness of a spring wheat variety (cv. Kohdasht) to cold from a proteomic point of view, in order to unravel the molecular mechanisms helping a cold sensitive variety to survive exposure to suboptimal temperatures. Plants were grown at 20 or 4 C until entering the reproductive stage and a cross-comparison on the leaf proteomes was performed. Quantitative analyses on protein alterations occurring upon low temperature exposure showed a reinforcement in ascorbate recycling (dehydroascorbate reductase, ascorbate peroxidase) and protein processing (proteasome subunit, cysteine proteinase), as well as the accumulation of the enzyme devoted to tetrapyrrole resynthesis (glutamate semialdehyde aminomutase). In contrast, among proteins down-regulated after cold stress, we could identify some key Krebs cycle enzymes (isocitrate dehydrogenase, malate dehydrogenase), together with many photosynthesis-related proteins (oxygen-evolving complex proteins, ATP synthase subunits, ferredoxin NADPH oxidoreductase, and some Calvin cycle enzymes). Physiological and biochemical parameters (such as shoot apex dissection, chlorophyll, proline and sugar content determination) sustained proteomics findings allowing the present research to contribute to the current knowledge on those long-term responses which may be crucial to stress adaptation under field conditions. Lello Zolla Full Professor Department of Environmental Sciences University of Tuscia Proteomics and Genomics Lab Largo dell Università snc Viterbo, Italy [email protected] tel fax

23 5.O.3 PROTEOMIC APPROACH TO CHARACTERIZE HOMOZYGOUS CITRUS CLEMENTINA HORT. EX TAN. GENOTYPES Maria Antonietta Germanà a*, Selenia Messina b, Gianluca Di Cara b, Nadia Ninfa Albanese b, Alessia Pivetti b, Maria Rita Marabeti b, Ida Pucci-Minafra b adipartimento DEMETRA Università degli Studi di Palermo, Palermo, Italy. bdipartimento di Oncologia Sperimentale e Applicazioni Cliniche, Palermo, Italy Tri-haploids have been regenerated through pollen embryogenesis (specifically, by anther culture) of Citrus clementina Hort. ex Tan., cv. Nules (Germanà et al., 2005). Two of them, (HOMO 1 and HOMO 2), have been acclimatized and grafted in vivo in Proteomic approach, allowing simultaneous separation and identification of hundreds of proteins in the same 2D-electrophoretic gel, permits the comparison of the proteome profiling in different samples. The most critical step in any proteomics study is sample extraction and preparation (Ramu et al., 2004) and, often, the electrophoretic separation of proteins from plant tissue extracts is complicated by proteases and materials that severely interfere with downstream protein separation and analysis (Tsugita and Kamo, 1999). Although there are some groups working on citrus proteomic profile, it is still poorly characterized, because there are many difficulties in the extraction of proteins. In this research, proteins have been extracted, trough the standardization of experimental methods, from leaves of homozygous and heterozygous citrus plants and separated by 2D-electrophoresis analysis, with the aim of achieve the higher number of quantitative and qualitative information about leaves proteins. High quality leaf proteomic maps obtained from the two homozygous regenerants have been analyzed and compared with that one of the heterozygous mother plant on the basis of the presence/ absence of spots. Notable differences in the protein expression have been observed in all the three genotypes. Prof. Maria Antonietta Germanà Dipartimento DEMETRA Università degli Studi di Palermo Viale Delle Scienze, Palermo -Italy [email protected] Tel Fax: Cell References: Germanà, M.A., Chiancone, B., Lain, O., Testolin, R. Anther culture in Citrus clementina: a way to rigenerate tri-haploids. Australian Journal of Agricultural Research. 2005, 56, Ramu, S., Saravanan J., K., Rose, C. A critical evaluation of sample extraction techniques for enhanced proteomic analysis of recalcitrant plant tissues. Proteomics. 2004, 4, Tsugita, A., Kamo, M.. 2-D Electrophoresis of Plant Proteins. Methods Mol. Biol. 1999, 112,

24 1.1 PROTEOMIC AND FUNCTIONAL ANALYSIS OF HUMAN, BOVINE AND CAPRINE MILK FAT GLOBULES Stefano Spertino 1, Chiara De Angelis 1, Valentina Cipriani 1, Gabriella M. Giuffrida 2, Maria Cavaletto 1 1 DISAV Dip Scienze dell Ambiente e della Vita Università del Piemonte Orientale A. Avogadro, Alessandria, Italy 2 CNR - Istituto di Scienze delle Produzioni Alimentari, Torino, Italy Lactating mammary gland secrete triacylglycerols in the form of Milk Fat Globules (MFGs) coated with a cellular membrane called the Milk Fat Globule Membrane (MFGM). MFGs have many biological properties due to both proteins associated with the MFGM (1-4% of total milk protein content) and lipid molecules, involved in various cellular processes and defense mechanisms in the newborn (1). Biochemical characterization of MFGM was achieved by proteomic analysis, while the MFGs functional role was investigated employing CaCo2 cells, in order to evaluate their nutraceutical properties. Milk samples from different sources were centrifuged for preparing MFG, MFGM proteins were extracted from MFG by methanol/chloroform precipitation. The resulting proteins were separated by 2DE, trypsin digested and identified by NanoLC ESI-Q-TOF MS/MS analysis. This approach led to the identification of major MFGM proteins (lactadherin, adipophilin, butyrophilin, perilipin, polymeric Ig receptor) and revealed the presence of some other minor MFGM components. Proteomics tools were employed to the characterization of N-glycosilation of MFGM proteins; the composition of released N-linked sugars was analyzed in MALDI-TOF MS showing that human milk is a rich source of complex oligosaccharides while their presence is lower in bovine and caprine milk. The functional analysis was performed by a 48-hours treatment of CaCo2 cells with crescent concentrations of MFG. The effect on vitality was evaluated by Neutral Red Uptake Assay showing a positive implication on cellular growth. These results show a potential nutraceutical role for the whole MFG, suggesting a possible employment in milk formula preparation. Maria Cavaletto Prof. DISAV- Dip. Scienze dell Ambiente e della Vita Univ. Piemonte Orientale A.Avogadro Viale T. Michel, Alessandria, Italy [email protected] tel fax This work was supported by Regione Piemonte grant (Ricerca sanitaria finalizzata n DA20.01) (1) Cavaletto, M. et al., Advances in Experimental Medicine and Biology, 606, (2008) 1.2 MALDI-TOF MS PROTEOMIC PHENOTYPING BOOSTS PATIENT-TAILORED PROTOCOLS IN DIAGNOSTIC MYCOLOGY Federica Del Chierico a, Andrea Masotti b, Manuela Onori a, Ersilia Fiscarelli c, Leopoldo Dimiziani d, Donato Menichella a, Andrea Urbani e,f and Lorenza Putignani a aunity of Microbiology, Bambino Gesù Children's Hospital and Research Institute, Piazza Sant Onofrio 4, 00165, Rome, Italy; bgene Expression-Microarrays Laboratory, Bambino Gesù Children's Hospital and Research Institute, Piazza Sant Onofrio 4, Rome, Italy; cunit of Microbiology of Cystic Fibrosis, Department of Pathology, Children s Hospital and Research Institute Bambino Gesù, Piazza Sant Onofrio 4, Rome, Italy; dbruker Daltonics s.r.l., Via Cluentina 26, 62100, Macerata, Italy; edepartment of Internal Medicine, University of Rome Tor Vergata, Rome, Italy, feuropean Center Brain research, IRCCS-Fondazione S. Lucia, Roma, Italy. Pediatric populations are currently at high risk for mould opportunistic infections due to high impact of changes in medical, intensive care and organ transplantation practices. Identification (ID) of the increasing diversity of fungal pathogens by conventional methods is often difficult, time-consuming and frequently, for unusual fungi, inconclusive. These constraints have led us to design and set-up an analytical MALDI-TOF MS-based assay to identify the most isolated and emerging/uncommon fungi encountered in daily microbiological efforts. The aim was achieved by generating a dedicated engineered library of reference spectra from solid Sabouraud agar (SDA), used in the diagnostic ID workflow. A growth time-course at 30 C was performed for 22 morphotypes belonging to 10 different genera and 2 Phyla (the Ascomycetes Aspergillus, Emericella, Fusarium, Geosmithia, Neosartorya, Penicillium, Pseudallescheria, Scedosporium, Talaromyces and the Basidiomycota Fomitopsis) at 48, 72, 96 and 120 hour points to address the best conditions for fully recovery of multihyphal structure producing either conidia or asci, depending on morph status, and to address spectra reproducibility. The 120 hour point was selected for generating the best morphotype spectra, based on correlation by unsupervised hierarchical clustering (Pearson similarity measure); fingerprinting classification by statistical Wilcoxon/Kruskal-Wallis test; classification robustness by recognition capability (RC) and cross-validation (CVA), achieved by Genetic Algorithm (GA), Support Vector Machine (SVM), Supervised Neuronal Network (SNN) and Quick Classifier (QC) model generation. Proteome profiling-based assay may be an optimal diagnostic approach to overcome traditional ID methods, to encompass multiple fungal genera, to reflect the variety of morph-types, growth phase and fungal microenvironment. Federica Del Chierico, Post-doctoral Research Fellow Microbiology Unit, Immunology Research Area, Children Hospital and Research Institute Piazza Sant Onofrio, 4, Rome, Italy [email protected] Phone: Fax:

25 1.3 GROWTH-PHASE RELATED PROTEOMICS CAN ADD NEW INFORMATION TO THE DIFFERENT CATABOLIC ATTITUDE OF A. RADIORESISTENS S13 TOWARDS PHENOL AND BENZOATE. Paolo Fattori a, Cristina Barello b, Maria Gabriella Giuffrida b, Carlo Giunta a and Enrica Pessione a a Dipartimento di Biologia Animale e dell Uomo. Università di Torino. Italy b ISPA-CNR, Bioindustry Park, Colleretto Giacosa (TO). Italy Acinetobacter radioresistens S13 is a gram-negative strain very efficient in Phenol degradation and therefore with a high potential in bioremediation. In spite of their structural similarity, Benzoate is metabolized slower and in lower biomass yield as compared to Phenol. The present proteomic investigation performed at different points (Early Exponential, Middle Exponential, Late Exponential and Stationary phase) of the bacterial growth-curve allowed to explain, in part, the metabolic differences observed. First of all Benzoate-grown bacteria show less time-course variations for the most interesting proteins considered (biosurfactants, energy metabolism enzymes) and express proteins involved in uptake (BenP) and extrusion (TonB) of Benzoate, absent in phenol grown cells. Furthermore the IsoB isoform of the Catechol 1,2 dioxygenase is biosynthesized later than IsoA and also this IsoB is absent in Phenol-cultures. Phenol, conversely, induces more time-course variations expecially for what concerns biosurfactant production, energy metabolism enzymes, stress proteins and ph homeostasis proteins; the late exponential phase being the time of maximum expression. The role of the detected proteins in confirming the different metabolic fate of Phenol and Benzoate is discussed. Paolo Fattori, PhD Università degli Studi di Torino Dipartimento di Biologia Animale e dell Uomo Via Accademia Albertina, 13, Torino, Italia. [email protected] Phone Fax RND EFFLUX TRANSPORTER GENE DELETION IN BURKHOLDERIA CENOCEPACIA : A PROTEOMIC ANALYSIS Tania Gamberi a, Maria Cristiana Papaleo b, Silvia Rocchiccioli c, Lorenzo Citti c, Renato Fani b, Alessandra Modesti a adepartment of Evolutionary Biology, University of Florence, Firenze, Italy bdipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Firenze, Italy cistituto di Fisiologia Clinica-CNR, Pisa, Italy Burkholderia cenocepacia is representative of a problematic group of cystic fibrosis pathogens. Eradication of B. cenocepacia is very difficult being the antimicrobial therapy ineffective due to its high resistance to clinically relevant antimicrobial agents1. RND (Resistance-Nodulation- Cell Division) efflux pumps are known to be mediators of multidrug resistance in Gram-negative bacteria2. Since the significance of the RND efflux systems in B. cenocepacia has been partially determined, the aim of this work was to analyze mutants of B. cenocepacia J2315 impaired in BCAL (RND-4) and BCAM (RND-9) pumps, and assess their role in the efflux of toxic compounds. A previous Phenotype MicroArray and a transcriptome were performed on mutants deleted individually in RND-4 and RND-9 (named D4 and D9), and a doublemutant in both efflux pumps (named D4-D9) revealing that motility and chemotaxis-related genes were up-regulated in both D4 and D4-D9 strains3. In order to confirm these data we performed a proteomic analyses of the three B. cenocepacia mutants using two-dimensional gel electrophoresis and MALDI-TOF/TOF Mass Spectrometry. We compared the proteome profile of the three mutants to that of the wild type strain J2315. As expected the majority of differences were found in the doublemutant (D4-D9) proteome. Nowadays we have concluded the analyses on RND-4 mutant proteome. This analyses pointed out 122 variations compared to wild type. Spots were extracted from 2-DE gels to be identified by MS. These results indicate that in B. cenocepacia RND pumps play a wider role than just in drug resistance, influencing phenotypic traits important for pathogenesis. Corresponding Author. Tania Gamberi, PhD Dipartimento Scienze Biochimiche Università degli Studi di Firenze Viale Morgagni 50, Firenze, Italy [email protected] Phone Fax References [1] Mahenthiralingam E, Baldwin A, Dowson CG. Burkholderia cepacia complex bacteria: opportunistic pathogens with important natural biology. J Appl Microbiol. (2008) 104: [2] Hasdemir U. The role of cell wall organization and active efflux pump systems in multidrug resistance of bacteria. Mikrobiyol Bul. (2007) 41: [3] Bazzini S, Udine C, Sass A, Pasca MR, Longo F, Emiliani G, Fondi M, Perrin E, Decorosi F, Viti C, Giovannetti L, Leoni L, Fani R, Riccardi G, Mahenthiralingam E, Buroni S. Deciphering the Role of RND Efflux Transporters in Burkholderia cenocepacia. PLoS One. (2011) 6:e18902.

26 1.6 PROTEOMICS TO EVALUATE FERTILITY LEVEL OF BULLS SEMEN FROZEN WITH THREE DIFFERENT EXTENDERS Alessandro Gaviraghi1, Alessio Soggiu2, Andrea Galli3, Cristian Piras4, Pamela Bonaccini5, Hany A. Hussein1, Luigi Bonizzi1, Paola Roncada3 1DIPAV, Facoltà di Medicina Veterinaria, Università degli Studi di Milano, Milano; 2Università degli Studi di Cagliari, Dipartimento di Neuroscienze B.B. Brodie, Cagliari; 3 Istituto Sperimentale Italiano L. Spallanzani, Milano; 4 Università degli studi di Sassari, Dipartimento di Scienze Zootecniche, Centro NanoBiotecnologie, Sassari; 5Dipartimento di Scienze Cliniche Veterinarie, Facoltà di Medicina Veterinaria, Università degli Studi di Padova, Padova. In dairy cattle breeding, herd reproductive management is the main topics and affects a large part of general costs. Much progress has been made in cattle management to increase female fertility, while the studies and activities are still scarce to increase the bull reproductive efficiency. Some studies have shown that a significant percentage of reproductive failure is attributable to semen quality and not to cow problems (1). In order to fertilize the oocyte, each spermatozoa must present motility, active mitochondria, intact membranes and a nucleus capable of proper decondensation and reorganization (2). In this study, proteomic analysis was applied to compare the sperm protein expression profile from bulls with a different level of fertility. Three different commercial extenders were used in this study. Image analysis allowed the differentiation of the different extenders used for freezing of bovine semen. Also, comparative proteomic analysis shown that expression of several proteins is related to high and low fertility. Their physiological functions or biochemical properties sustain plausible implications in sperm functionality defects, where lack or overexpression can compromise sperm chances to reach and fertilize the oocyte. For this reason, proteomics of sperm can be a valuable tool to identify protein changes related to fertility. 2DE-based proteome analysis is a powerful tool to characterize expression of spermatozoa proteins related to high and low fertility rate. Alessandro Gaviraghi, Ph.D DIPAV, Facoltà di Medicina Veterinaria, Università degli Studi di Milano Via Celoria 10, Milano. [email protected] Phone: References 1.DeJarnette J..M.., Marshall, C.E., Lenz R.W., Monke D.R., Ayars W.H., Sattler C.G. Sustaining the Fertility of Artificially Inseminated Dairy Cattle: The Role of the Artificial Insemination Industry. Journal of Dairy Science (E. Suppl.), E93 E Graham J.K., Moce E. Fertility evaluation of frozen/thawed semen. Theriogenology , PROTEOMIC CHARACTERIZATION OF HUMAN MILK PROTEINS DURING A SIXTY-DAYS LACTATION PERIOD Alessandro Gaviraghi1, Alessio Soggiu2, Cristian Piras3, Luigi Bonizzi1, Carlo Agostoni4, Paola Roncada5 1DIPAV, Facoltà di Medicina Veterinaria, Università degli Studi di Milano, Milano; 2Università degli Studi di Cagliari, Dipartimento di Neuroscienze B.B. Brodie, Cagliari; 3Università degli studi di Sassari, Dipartimento di Scienze Zootecniche, Centro NanoBiotecnologie, Sassari; 4Università degli Studi di Milano, Fondazione IRCCS Ca' Granda-Ospedale Maggiore Policlinico, Milano; 5Istituto Sperimentale Italiano L. Spallanzani, Milano Human milk is a rich source of bioactive proteins that support the growth and development of the newborn. Although the major components of the protein fraction in human milk have been studied, the expression and relative abundance of milk proteins during the different periods of lactation have received limited attention. The aim of this project is to analyze the human milk proteome at two days, seven days, fifteen days and sixty days of lactation in order to identify the best composition for the development of an infant formula milk powder as similar as possible to human milk. As result was founded a gradual increase in the concentration of immunoglobulins heavy chain and of albumin from two to sixty days of lactation. Moreover a significant increase of lactoferrin, α-lactalbumin and β-casein was found in the first week of lactation, from day seven to day sixty this values were found to be stable. In order to have a reference map for the evaluation of infant formula milk powder, a master 2D map was obtained from the analysis of all eighteen samples of each time-point. Image analysis also highlighted a subunit of β-casein that showed a gradual decrease at sixty days of lactation and other two proteins differentially expressed. These results enhance our knowledge about the complexity of the human milk proteome, and are useful to be compared with the protein content of infant formula milk powder. Alessandro Gaviraghi, Ph.D DIPAV, Facoltà di Medicina Veterinaria, Università degli Studi di Milano Via Celoria 10, Milano. [email protected] Phone: Work supported by Heinz. Special Acknowledgements to Fabio Mosca, Unità di Neonatologia e Terapia Intensiva Neonatale, Ospedale Maggiore Policlinico - Mangiagalli e Regina Elena IRCCS Milano Acknowledgements: The authors are grateful to ANAFI for ERCR data. Work supported by PROZOO project, W.U. ISILS

27 1.9 EXOPROTEOME OF THE PROBIOTIC LACTOBACILLUS REUTERI LB2 BM: WHAT HAPPENS IN THE HOST-PROKARIOTE INTERFACE. Cristina Lamberti1, Erika Mangiapane1, Marco Cietto1, Giuliana Lo Bianco1, Ritva Virkola2, Timo Korhonen2, Enrica Pessione1. 1Dipartimento di Biologia Animale e dell Uomo, Università di Torino, Italy 2Departments of General Microbiology and Medical Chemistry, University of Helsinki, Finland 1.10 A COMBINED ICP-MS AND CLASSICAL PROTEOMIC APPROACH TO ANALYZE SELENIUM UP-TAKE BY A PROBIOTIC LACTOBACILLUS REUTERI Erika Mangiapane a, Eugenio Galano b, Cristina Lamberti a, Alessandro Pessione a, Angela Amoresano b, Pietro Pucci b, Enrica Pessione a a University of Torino, Department of Human and Animal Biology, Torino, Italy b University of Napoli, Department of Biochemistry and Organic Chemistry, Napoli, Italy Lactobacillus reuteri Lb2 BM, patented for its probiotical traits, is a very promising strain also for its ability to organicate selenium (Lamberti et al., 2011). The present study was aimed to shed light on the proteins secreted extracellularly in order to assess strain safety (the main pathogenic potential lies in the secretome) and adhesion capabilities. Of the 21 identified proteins 10 are sugar-metabolism enzymes, 4 are cell-wall involved proteins and 3 are stress proteins. Among these proteins, some are known for having adhesive capabilities for collagen (trigger factor) and for fibronectin (GAPDH and elongation factor Tu). Phenotypic investigations reveal that L. reuteri Lb2 BM is able to adher to collagen, fibronectin and laminin and that adhesion is maximum at ph 4. On the contrary adhesion experiments performed at higher ph (ph 8) show a significant decrease of the adhesive potential. On the basis that the adhesion mechanism was chiefly linked to surface-bound GAPDH, this enzyme has been searched on both surface and extracellular extracts at different phs. The presence of high amounts of surface-bound proteins at ph 4 and of free extracellular GAPDH at ph 8 confirm the hypothesis. Capability to bind and activate PLG has also been evaluated demonstrating a weak PLG-binding capability. All these data confirm the safety of the strain in study and underline importance of exoproteome analysis for better understanding what happens in the host-prokariote interface. Cristina Lamberti Cristina Lamberti, PhD, Dipartimento di Biologia Animale e dell Uomo, Università degli Studi di Torino, via Accademia Albertina 13, Torino, Italia, [email protected] tel: fax: Lamberti C., Mangiapane E., Pessione A., Mazzoli R., Giunta C., Pessione E. (2011) Proteomic characterization of a seleniummetabolizing probiotic Lactobacillus reuteri Lb2 BM for nutraceutical application. Proteomics, Epub aehad of print. Selenium (Se) has recently received particular attention [1] since it is required for many cell processes and enzymes, although it becomes deleterious at high doses. Se deficiency is associated with poor health, cardiovascular diseases and it is also responsible for oxidative stress, since it is present as selenocysteine in the active site of glutathione peroxidase [2, 3]. The use of Se-enriched lactobacilli is an interesting approach to solve selenium-deficiency: these microorganisms are able to convert inorganic selenium into more bio-available organic forms, introducing it into proteins (selenoproteins); so, if added to ice-creams or yogurt, organic Se forms are released at host gut level. In this study we focused our attention on Lactobacillus reuteri LB2 BM, isolated from human fecal samples. In order to investigate the time and the dose of the internalized selenium during its growth in a MRS medium fortified with sodium selenite, a microwave assisted digestion procedure, used for the dissolution of biological samples, followed by ICP-MS analyses, have been performed on samples collected at different times of the growth curve. Moreover a two-dimensional electrophoresis approach followed by MALDI-TOF/TOF mass spectrometry was performed for studying the membrane-cell wall enriched fraction, in order to eventually highlight an over-expression of some membrane transporters in the presence of selenium. This analysis revealed 12 differentially expressed spots: particular attention must be addressed to some classical cytosolic proteins (i.e. putative elongation factor Tu, α-enolase) that, when present in another cellular district (cell wall in this case), are defined moonlighting proteins. Erika Mangiapane PhD student Università degli studi di Torino, Dipartimento di Biologia Animale e dell Uomo Via Accademia Albertina 13, 10123, Torino [email protected] Phone: Fax References: [1] Fisinin, V. I., Papazyan, T. T., Surai, P. E., Producing seleniumenriched eggs and meat to improve the selenium status of the general population. Crit. Rev. Biotechnol. 2009, 29, [2] Rayman, M. P., Food-chain selenium and human health: emphasis on intake. Br. J. Nutr. 2008, 100, [3] Combs, G. F., Selenium in global food systems. Br. J. Nutr.2001, 85,

28 1.11 A PROTEOMIC APPROACH TO SEARCH BIOMARKERS USEFUL FOR IN VIVO SCRAPIE DIAGNOSIS Maria Mazza a, Chiara Guglielmetti a, Francesca Martucci a, Marianna Pagano a, Paola Marconi b, Gianfranco Santagada c, Pasquale Troiano c, Maurizio Bruschi d, Giovanni Candiano d, Pier L. Acutis a a CEA - Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d Aosta, Torino, Italia b Istituto Zooprofilattico Sperimentale del Lazio e della Toscana, Firenze, Italia c Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata, Foggia, Italia d Laboratorio di Fisiopatologia dell Uremia, Ospedale Pediatrico G. Gaslini, Genova, Italia Scrapie in sheep and goats, like bovine spongiform encephalopathy (BSE) and human Creutzfeldt Jakob disease (CJD), belongs to the transmissible spongiform encephalopathies (TSEs), a group of fatal and neurodegenerative disorders, also defined as prion diseases. These diseases are associated with the conversion of the normal prion protein (PrPC), widely expressed in the central nervous system, into a partially protease-resistant isoform (PrPSc) [1]. The susceptibility of sheep to scrapie is known to be linked to polymorphisms of the PrP encoding gene (PRNP) at position 136, 154 and 171 [2]. The diagnosis of scrapie and other TSEs is currently based on the post-mortem detection of the PrPSc in the nervous system. Several attempts have been made to research PrPSc in lymphoid tissue biopsies or biological fluids [3,4] but they are not very accurate antemortem methods to diagnose TSEs, so different studies have been performed to identify alternative biomarkers in accessible tissues or body fluids. With this purpose we carried out a study on the sera of sheep with natural scrapie by two dimensional gel electrophoresis. Ovine serum samples were collected from four different Italian naturally scrapie-affected flocks. Twenty animals were chosen, ten positive susceptible and ten negative resistant or semi-resistant sheep, coupled by age, sex and breed. Serum samples were subjected to 2D analysis, using different protocols. To date, eight serum samples were analysed. From the preliminary data analysis no significant differences were present between positive and negative animals. The analysis of all the sampled animals, also with more sensitive revelation methods, will better clarify whether scrapie status indirect markers can be detected and identified. References: [1] Prusiner S.B. "Molecular biology of prion diseases. Science, 1991, 252: [2] Baylis M., Goldmann W. The genetics of scrapie in sheep and goats. Current Molecular Medicine, 2004, 4 (4): [3] Schreuder B., van Keulen L., Vroman, M., Langeveld J. and Smits M. Tonsillar biopsy and PrPSc detection in the preclinical diagnosis of scrapie. The Veterinary Record, 1998, 142(2 1): [4] Rubenstein R., Chang B., Gray P., Piltch M., Bulgin M.S., Sorensen- Melson S. and Miller M.W. A novel method for preclinical detection of PrPSc in blood. J. Gen. Virol., 2010, 91: Corresponding author: Dr. Maria Mazza CEA - Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'aosta, Via Bologna 148, Torino, Italy [email protected] Phone: Fax: IMMUNOGENIC PROTEINS AS PUTATIVE TARGETS FOR MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS DETECTION IN CATTLE Cristian Piras1, Alessio Soggiu2, Luigi Bonizzi3, Gian Franco Greppi1, Norma Arrigoni4, Paola Roncada5 1Università degli studi di Sassari, Dipartimento di Scienze Zootecniche, Centro NanoBiotecnologie, Sassari; 2Università degli Studi di Cagliari, Dipartimento di Neuroscienze B.B. Brodie, Cagliari; 3DIPAV, Facoltà di Medicina Veterinaria, Università degli Studi di Milano, Milano; 4Centro di Referenza Nazionale per la Paratubercolosi, IZSLER Sezione Diagnostica di Piacenza 5Istituto Sperimentale Italiano L. Spallanzani, Milano Johne s disease occurs worldwide and it has been estimated that 5 to 20% of cattle are infected causing enormous economic losses. The infection is due to the direct contact with infected excrements or to the administration of infected milk or water. MAP is ubiquitous and it can be found in soil and water of the infected farms, but actually there is no available method to find it. Extra sensitive method for MAP detection is strongly required; several diagnostic methods have been proposed [1, 2], but nothing seems useful to detect small amounts of MAP in matrices like soil, milk and water. Aim of this work is to evaluate the cytoplasmic and membrane MAP proteome to find potential protein targets for developing a specific biosensor. As results it has been developed a very efficient extraction method of both cytoplasmic and membrane proteins that is able to detect more than 1000 proteins in our 2D maps. After 2D immunoblotting against serum of infected cattle we founded several immunogenic proteins useful as putative targets for developing a biosensor. In particular one is really interesting because of its similarity with a previously described immunogenic protein, the MAP2121c, that is a 35 kda membrane protein with a strong immunogenic activity and specificity [3]. In conclusion an experiment demonstrated, according with previous findings, that, because of its abundance and MAP specificity, MAP2121c can be used as possible candidate for developing a biosensor. Cristian Piras, Ph.D Università degli studi di Sassari, Dipartimento di Scienze Zootecniche, Centro NanoBiotecnologie, Sassari [email protected] Phone: Bech-Nielsen, S., et al., Diagnostic accuracy of a Mycobacterium phleiabsorbed serum enzyme-linked immunosorbent assay for diagnosis of bovine paratuberculosis in dairy cows. J Clin Microbiol, (3): p Collins, M.T., et al., Crossed immunoelectrophoretic analysis of Mycobacterium paratuberculosis. APMIS, (1): p Bannantine, J., et al., Development and use of a partial Mycobacterium avium subspecies paratuberculosis protein array. Proteomics, (3): p Acknowledgements This work was supported by a fellowship financed by RAS (Regione Autonoma della Sardegna, PO Sardegna FSE L.R. 7/2007 Promozione della ricerca scientifica e dell innovazione tecnologica in Sardegna )

29 1.13 PROTEOME ANALYSIS OF POST-MORTEM CHANGES IN BOVINE LONGISSIMUS DORSI BY TWO-DIMENSIONAL (2-DE), P-DIMENSIONAL (2-PE) ELECTROPHORESIS AND RANKING-PCA Michele Menini a, Rita Polati a, Elisa Robotti b, Luca Fasolato c, Filomena Montemurro c, Emilio Marengo b, Renato Millioni d, and Daniela Cecconi a a Dept. of Biotechnology, Mass Spectrometry and Proteomics Lab, University of Verona, Italy b Dept. of Environmental and Life Sciences, University of Eastern Piedmont, Italy c Dept. of Public Health, Comparative Pathology, and Veterinary, University of Padua, Italy d Dept. of Clinical and Experimental Medicine, Chair of Metabolism, University of Padua, Italy To study early post-mortem changes in muscle tissue from bovine Longissimus dorsi at 0, 12 and 26 days after slaughter we analyzed the proteome by two-dimensional (2-DE), as well as, by P-dimensional electrophoresis (2-PE). This new method, called 2-PE, takes advantage of the presence of a SDS-PAGE step with a radial electric field instead of a parallel one. Since spots with close but not equal pi are moved by diverging lines of forces, their resolution increases during the radial separation, by a factor proportional to the migration distance. Moreover reproducibility between replica gels is enhanced. Ranking principal component analysis (Ranking-PCA) was used to analyse the protein patterns obtained by 2-DE and 2-PE in order to select spots that were significantly different at the three time-points. Selected proteins were identified by nanohplc-chip Ion trap MS/MS. Among the modulated proteins we have identified stress proteins members of the small heatshock protein family (such as Hsp27 and HspB6), structural proteins (such as troponin T, fast and slow isoforms), and metabolic enzymes (such as GAPDH). Further studies will reveal the relevance of these proteins changes during post-mortem storage of bovine muscles to control better meat quality. "Corresponding Author" : Daniela Cecconi, PhD Mass Spectrometry & Proteomics Lab Department of Biotechnology, University of Verona, Strada le Grazie 15, Verona, Italy, phone: , fax: PROTEOMIC INVESTIGATION OF A NEW POSSIBLE OMEGA-OXIDANT BACTERIUM: A STUDY ON ACINETOBACTER RADIORESISTENS S13 Zapponi Michele, Fattori Paolo, Mazzoli Roberto, Pessione Alessandro, Giunta Carlo and Pessione Enrica Università degli Studi di Torino, Lab. Proteomica e Biochimica dei Microrganismi, DBAU In the present research we try to obtain biodegradable polymers from vegetable oils, low-cost industrial by-products that constitute a renewable source of fatty acids (FA). The limiting step of FA polycondensation is the lack of a second carboxyl or hydroxyl function in a terminal (ω) or subterminal (ω-1) position. We employed an aromatic-degrading Acinetobacter radioresistens S13 strain whose hydroxylating ability, via both mono- and di- oxygenases, has been previously proved [1]. The strain demonstrated to be able to metabolize aliphatic substrates (from C9 to C18), suggesting the presence of an ω-oxidative enzyme system. Then we tested growth on fatty acids, obtaining satisfactory results for C9 and C18: considering these results and the availability of industrial wastes, we chose pelargonic acid as the most interesting substrate. We set up a gas chromatography method to verify the consumption of carboxylic acids and the parallel formation of metabolites of interest, with the goal to determine the best conditions for comparative proteomic experiments. The first proteomic experiment was performed in order to compare protein expression with acetate (control) and pelargonate as sole carbon sources. After image analysis we identified 50 spots with significant expression variation. After that we evaluated the consumption of pelargonic acid when it was present as the sole carbon source and when it was supplemented with acrylic acid (β-oxidation inhibitor) and acetate as second carbon source. Results show complete metabolization of pelargonic acid despite the β-oxidation inhibitor: next proteomic experiments will be performed confronting these growth conditions. [1] Pessione et al., Eur. J. Bioch., 2003 Michele Zapponi Michele Zapponi PhD student Università degli Studi di Torino Lab. Proteomica e Biochimica dei Microrganismi, DBAU, Via Accademia Albertina 13, Torino [email protected] References [1] E. Marengo, E. Robotti, M. Bobba, F. Gosetti, The principle of exhaustiveness versus the principle of parsimony: a new approach for the identification of biomarkers from proteomic spot volume datasets based on Principal Component Analysis, Analytical and Bioanalytical Chemistry 397(1), 25-41, 2010 [2] E. Robotti, M. Demartini, F. Gosetti, G. Calabrese, E. Marengo, Development of a classification and ranking method for the identification of possible biomarkers in proteomics based on Principal Component Analysis and variable selection procedures, Molecular Biosystems 7(3), , 2011

30 1.16 PVL RELATED DIFFERENCES IN PROTEIN EXPRESSION PROFILE IN MRSA AND MSSA Hany A. Hussein 1, Alessandro Gaviraghi 1, Cristian Piras 2, Alessio Soggiu 3, Monica Monaco 4, Annalisa Pantosti 4, Marco Tinelli 5, Luigi Bonizzi 1, Paola Roncada 6 1 DIPAV, Facoltà di Medicina Veterinaria, Università degli Studi di Milano, Milano 2 Università degli Studi di Sassari, Dipartimento di Scienze Zootecniche, Centro NanoBiotecnologie, Sassari 3 Università degli Studi di Cagliari, Dipartimento di Neuroscienze B.B. Brosie, cagliari 4 Istituto Superiore di Sanità, Roma 5 U.O Malattie Infettive e Tropicali, Azienda Ospedaliera della Provincia di Lodi 6 Istituto Sperimentale Italiano L. Spallanzani, Milano Since its early discovery as an opportunistic pathogen, Staphylococcus aureus continues to be a major cause of mortality with a wide variety of clinical affections. Panton-Valentine Leukocidin (PVL) is a staphylococcal synergohymenotropic exotoxin (one of virulence factors) belonging to the pore-forming toxin family, induces lysis of host defense cells such as human neutrophils, monocytes, and macrophages, and recently been associated with necrotizing pneumonia. Although MRSA was considered a worldwide major threat, recent records demonstrated several clinical cases of staphylococci infections caused by MSSA with high prevalence of (PVL) + isolates. Hence, understanding mechanisms of both cell physiology and pathophysiology is necessary to contrast the diffusion of this pathogen, the aim of this project is to study the proteome of MSSA and MRSA with special emphasis on PVL + and PVL - strains to identify proteins that may be related to virulence using 2-D electrophoresis and mass spectrometry. Proteomic analysis revealed 8 differentially expressed proteins between MSSA (PVL - ) and MSSA (PVL + ) groups. Five of them showed over expression in MSSA (PVL) - while 3 proteins were over expressed in MSSA (PVL) +. These proteins were successfully identified by mass spectrometry. Focusing on the function of identified proteins, it was found that proteins overexpressed in PVL + are linked with catalysis of polypeptides and with energy production pathways. While the ones overexpressed in PVL are related to transcription pathways. Results can explain the reason because PVL + strains are more pathogenic than PVL -. Corresponding author: Hany A. Hussein DIPAV, Facoltà di Medicina Veterinaria, Università degli Studi di Milano. Via Celoria, 10 Milano [email protected] Phone:

31 2.2 ANALYSIS OF ENDOTHELIAL CELL SECRETOME IDENTIFIED PENTRAXIN-3 AS A NOVEL TARGET OF THE LIPID-LOWERING HMG-COA REDUCTASE INHIBITORS Sabrina Lento a, Maura Brioschi a, Valentina Bozzola a, Sabrina Galli a, Elena Tremoli a,b and Cristina Banfi a acentro Cardiologico Monzino I.R.C.C.S., Milan, Italy, bdepartment of Pharmacological Sciences, University of Milan, Italy. The clinical benefits of HMG-CoA reductase inhibitors (statins) are strongly related to their cholesterol lowering properties. However, because mevalonic acid, the product of HMG-CoA reductase reaction, is the precursor not only of cholesterol but also of nonsteroidal isoprenoid compounds, the inhibition of HMG-CoA reductase may result in pleiotropic effects. Indeed, statins can interfere with major events involved in the formation of atherosclerotic lesions, but these effects have not been fully elucidated. The analysis of endothelial cell secretome was performed by the use of the mass spectrometry based method (LC/MSE) for label-free quantitative proteomics, and identified PTX3, which belongs to the superfamily of acute-phase proteins pentraxins, as a novel pleiotropic target downregulated by statins. We demonstrate the importance of HMG-CoA reductase inhibition in the observed effects by showing the overcoming of the statins effects on PTX3 by the immediate product of HMG-CoA reductase, mevalonate, but not by cholesterol, the final product of the mevalonate pathway. To better understand the mechanisms involved, we further investigated the metabolites downstream of the mevalonate pathway. Only the addition of the isoprenoid intermediate GGPP, but not FPP, completely reverted statin effects, and consistently, the inhibitor of geranylgeranyltransferase I mimicked atorvastatin effect. This indicates that statin interfere with PTX3 by negatively interfering with the geranylgeranylation of some cellular proteins, with candidates being the Rho subfamily of monomeric GTPases. The addition of the Rac1/2 inhibitor and the specific silencing of Rac2 closely mimicked statin effect on PTX3, indicating that the Rac2 pathway plays a pivotal role in PTX3 regulation in endothelial cell. In conclusion, considering that PTX3 is endowed with activities that range from innate immune responses, inflammation to female fertility, extracellular matrix construction, angiogenesis, and cardiovascular diseases, the present findings indicate that statins can interfere with PTX3 functions. Funding: EC, FP6, LIFESCIHEALTH-contract n LSHM-CT PROCARDIS, grant from Italian Ministry of Health, Rome, Italy (Ricerca Corrente 2010 BIO 30), and it was also cofunded by 5x1000 Italian tax contribution. Corresponding author Cristina Banfi, PhD Centro Cardiologico Monzino IRCCS, Via Parea 4, Milano MI, Italy [email protected] Tel: ; Fax: ; 2.3 THE PROTEOME OF CARDIAC MICROPARTICLES Maura Brioschi a, Simona Scarpella a, Sabrina Galli a, Elena Tremoli a,b, Cristina Banfi a acentro Cardiologico Monzino I.R.C.C.S., Milan, Italy, bdepartment of Pharmacological Sciences, University of Milan, Italy. MPs are submicron vesicles shed from the cellular membrane upon activation of apoptosis. Most cells, including platelets, leucocytes, and endothelial cells, are capable of releasing membrane MPs in the circulation. MPs are characterized by their size (diameter ranging from 0.1 to 1 µm), the presence of externalized phosphatidylserine at their surface, and a specific antigenic profile reflecting their cellular origin. MP support coagulation by exposure of tissue factor, the initiator of coagulation in vivo. Moreover, MP may transfer bioactive molecules to other cells, thereby stimulating them to produce cytokines, cell-adhesion molecules, growth factors and TF, and modulate endothelial functions.numerous reports have highlighted changes in circulating MP levels in association with cardiovascular diseases, including stable and unstable CAD, as evidence of ongoing thrombosis, inflammation, cell injury, or apoptosis. It has been recently reported that high values of circulating CD31+/Annexin V+ MPs are associated with a worse clinical outcome, including an increased incidence of adverse cardiovascular and cerebral events. Furthermore, heart muscle injury during cardiopulmonary bypass surgery was associated with increased amounts of highly procoagulant cell-derived MPs in the pericardial blood. However, the cellular origin of these MPs is unknown. We thus postulate that also cardiomyocytes could be potential sources of circulating MPs. We therefore exposed cardiomyocytes to ischemia-hypoxia stress or to the cytokine TNFα. MPs were isolated from the cell culture media and analysed by the use of the mass spectrometry based method (LC/MSE) for label-free quantitative proteomics to gain a comprehensive characterization of the antigenic composition of MPs in both conditions. The analysis allowed the identification of a total of 514 proteins, among which 168 were differentially expressed: 68 uniquely expressed in hypoxia released MPs, 114 uniquely expressed in TNFα realeased MPs, 93 upregulated in hypoxia released MPs and 75 upregulated in TNFα realeased MPs. Gene Ontology analysis was performed in order to identify the overrepresented categories for biological processes and molecular function using the functional annotation clustering tool with David 6.7. In conclusion, this study allowed us to fully characterize the protein content of MPs released by cardiomyocytes exposed to different stimuli. Funding: EC, FP6, LIFESCIHEALTH-contract n LSHM-CT PROCARDIS; Italian Ministry of Health, Rome, Italy (Ricerca Corrente 2009 BIO 27). Corresponding author Cristina Banfi, PhD Centro Cardiologico Monzino IRCCS, Via Parea 4, Milano MI, Italy [email protected] Tel: ; Fax: ;

32 2.4 ERYTHROCYTES PRXII: OLIGOMERIC CHARACTERIZATION OF A CANDIDATE BIOMARKER OF OXIDATIVE STRESS DURING STORAGE UNDER BLOOD BANKING CONDITION. Barbara Blasi, Sara Rinalducci, Gian Maria D Amici, Lello Zolla Department of Environmental Sciences, University of Tuscia, Viterbo, Italy. During blood storage the erythrocytes undergo many changes, most of which at the membrane level. In particular they are subjected to oxidative stress-related phenomena that finally impair membrane structure. The individuation of proteins to be used as indicators of irreversible RBC injury is therefore of great interest. We have performed proteomic analysis of membranes during the storage period it used to last a bag of erythrocytes concentrates (42 days at the most). Changes in protein composition of RBC membranes were monitored by means of PAGE coupled with immunoblotting and MS analyses. During storage, a progressive migration of PrxII to the membrane was detected. This phenomenon was unequivocally related to oxidative stress, since storage of RBCs under anaerobic conditions showed a suppression of these protein recruitments to the membrane. Since it is well known that Prx is subjected to redox-induced structural transitions, we monitored its oligomeric state changes during storage by means of PAGE coupled with western blot. We found out that in fresh red cells Prx is present in 4 protein complexes in the cytosol while the storage brought to its presence only in three. At the same storage time, at the membrane level, we found PrxII in one complex. No PrxII was detected in fresh red cells membranes. The detailed analysis of these protein associations to the membrane of aged RBCs allowed PrxII to be suggested as a potential RBC oxidative stress marker for the sake of developing new approaches in quality assurance of blood components. Corresponding author: Lello Zolla Professor Department of Environmental Sciences University of Tuscia Largo dell Università snc, 01100, Viterbo [email protected] (+39) (39) DIFFERENTIAL PROTEIN EXPRESSION PROFILING IN HUMAN TEARS OF PATIENTS WITH OPHTHALMOLOGIC DISEASES BY DIFFERENT PROTEOMIC APPROACHES Damiana Pieragostino a,b1, Sonia Bucci b,c1, Luca Agnifili d, Vincenzo Fasanella d, Simona D Aguanno f, Marco Ciancaglini e, Leonardo Mastropasqua d, Andrea Urbani b,f, Carmine Di Ilio a,b, Paolo Sacchetta a,b and Piero Del Boccio a,b a Dept. of Biomedical Sciences, University G. d Annunzio of Chieti-Pescara Italy b Research Center on Aging (Ce.S.I), University G. d Annunzio of Chieti-Pescara Italy c Dept. of Neuroscience and Imaging, University G. d Annunzio of Chieti-Pescara Italy d Regional Center of Excellence in Ophthalmology University G. d Annunzio of Chieti-Pescara Italy e Ophthalmology Clinic, University of L'Aquila Italy f European Brain Research Institute - IRCCS Santa Lucia, Roma 1DP and SB contributed equally to this work. Primary open angle glaucoma (POAG) is the most common form of glaucoma, an optic neuropathy characterized by the loss of retinal ganglion cells and a typical atrophy of the optic nerve as consequences of elevated intraocular pressure and microcirculatory alteration. Pseudoexfoliation syndrome is characterized by the production of abnormal fibrillar extracellular material which accumulates in intraocular tissues, often leading to pseudoexfoliation glaucoma (PEXG), the most common type of secondary glaucoma. Currently, these ophthalmic diseases are poorly investigated and the diagnosis is often delayed. Tears are a source of nourishment for the eye and the study of tear proteome may offer useful information on the eye physiopathological condition, representing an ideal biological fluid for biomarker research. Our study fits into this context with the aim of protein characterization in tears of POAG and PEXG patients, using different proteomic techniques. We performed a comparative proteomic analysis by label-free LC-MSE of tears from POAG and PEXG patients, highlighting several proteins differently expressed between the two pathological conditions and control subjects and independently reconfirmed by monodimensional SDS-PAGE or linear MALDI-TOF/MS analysis. These screening investigation associated with bioinformatics analysis revealed that differently expressed proteins are involved in the inflammatory state and both innate and specific immune response. As a convergence of several strategies, this work allows to obtain important information from each clinical group studied and to confirm the results by independent analytical techniques. Hence, this work represents the beginning of a new, interdisciplinary research strategy on clinical, biochemical, proteomics and statistics fields. Sonia Bucci Department of Neuroscience and Imaging "G. d'annunzio" University Via dei Vestini 33, Chieti - Pescara, Italy [email protected] phone: Fax:

33 2.6 MOUSE TO HUMAN AUTOANTIBODY SIGNATURE IN PANCREATIC DUCTAL ADENOCARCINOMA Michela Capello1, Paola Cappello1, Federica C. Linty1, Sammy Ferri-Borgogno1, Roberto Chiarle2, Aldo Scarpa3, Paolo Pederzoli4, Paola Nisticò5, Michele Milella6 and Francesco Novelli1 1Center for Experimental Research and Medical Studies (CeRMS) and University of Turin, Turin, Italy 2Department of Biomedical Sciences and Human Oncology University of Turin, Turin, Italy 3Department of Pathology, University of Verona, Verona, Italy 4Department of Surgical and Gastroenterological Sciences, University of Verona, Verona, Italy 5Laboratories of Immunology, Regina Elena National Cancer Institute, Rome, Italy 6Medical Oncology, Regina Elena National Cancer Institute, Rome, Italy BACKGROUND & AIMS: Pancreatic Ductal Adenocarcinoma (PDAC) is an highly aggressive malignancy with a 5-year survival rate of only 5%. Biomarkers for its early detection are lacking. Refined genetically engineered mouse (GEM) models of PDAC faithfully recapitulate the human disease. In this study we have used a well-characterized GEM model of PDAC to identify antigens that elicit an early humoral response as diagnostic tools. METHODS AND RESULTS: Proteins from mouse PDAC cell line were separed by two-dimensional electrophoresis (2-DE) and the serum reactivity of PDAC GEM mice at early and advanced stages of tumor development was tested by Western blot (WB) analysis and compared to matched controls. Spots specifically recognized by PDAC sera were identified by MALDI-TOF MS and corresponded to two nuclear and six cytoskeletal proteins. Autoantibodies against these proteins were present already at the earliest stages of tumor development. According to microarray data, the mrna of some on these proteins was over-expressed in PDAC compared to normal pancreas. The autoantibody signature was validated by 2-DE WB in a set of advanced and resectable PDAC patients and controls (healthy subjects, non-pdac tumor patients, chronic pancreatitis and autoimmune diseases patients) sera. One cytoskeletal protein showed the higher frequency of autoantibodies in GEM mice with early disease and in resectable PDAC patients. The ELISA detecting autoantibodies against this protein showed high sensitivity and specificity. CONCLUSIONS: Our results indicate that GEM models of PDAC, in combination with SERological Proteome Analysis, provide a useful strategy to identify proteins able to induce an early autoantibody response in PDAC. To confirm their diagnostic value, these antigens will be validated by an handy assay in a large cohort of PDAC patients. Francesco Novelli, PhD Center for Experimental Research and Medical Studies (CeRMS) San Giovanni Battista Hospital, Via Cherasco 15, Turin, Italy Tel: Fax: [email protected] 2.7 AN ALTERNATIVE APPROACH FOR THE IDENTIFICATION AND QUANTIFICATION OF POTENTIAL PROSTATE CANCER BIOMARKERS. Francesca Casadonte1, Anna Napoli2, Donatella Aiello2, Rocco Damiano1, Rosa Terracciano1, Giovanni Sindona2 and Rocco Savino1. 1 Department of Experimental and Clinical Medicine G. Salvatore, University of Catanzaro Magna Græcia, Italy. 2 Department of Chemistry, Calabria University, Italy. Prostate cancer (PCa) is the most common noncutaneous cancer and the second leading cause of cancer-related death among men in Western countries (1). In the early diagnosis of the disease a crucial role is played by the prostate-specific antigen (PSA), whose value increases in the presence of cancer (> 4 ng/ml). Recent studies show that indeed there is no safe limit value below which it can be assumed that prostate cancer is absent (2). Moreover, the value of PSA has low specificity in differentiating malignant from benign conditions, resulting in inappropriate diagnosis and overtreatment (3). The identification of new biomarkers that allow early detection of cancer, distinguishing between indolent and aggressive form, and are specific to PCa and PCa recurrence are an urgent, unmet medical need. The aim of this project is the analysis of differential proteome through the study of tumoral and normal prostate tissue of patients with PCa, to identify potential tissue biomarkers for PCa. Prostate tissue specimens from patient with PCa were collected and analyzed to identify proteomics-based biomarkers useful for prostate cancer diagnosis. We analyzed the tumoral and non tumoral tissue from the same individuals. The hydrosoluble tissue protein extraction and protein chemical fractionation were performed to study the sub-proteome components. Moreover, to remove the high abundant proteins, as albumin, immunoglobulin and transferrin, we developed an alternative methods compatible with mass-spectrometry analysis. The low molecular weight proteins were proteolitically digested with trypsin, labeled and analyzed by MALDI-TOF-TOF Tandem Mass Spectrometer to study quantitative proteins expression. Francesca Casadonte Postdoc University of Catanzaro Magna Græcia Viale Europa, Catanzaro [email protected] Phone: Fax: References: 1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thu MJ CA Cancer J Clin; 58; Schröder FH, Carter HB, Wolters T, van den Bergh RC, Gosselaar C, Bangma CH, Roobol MJ Eur Urol. 53(3): Byrne JC, Downes MR, O'Donoghue N, O'Keane C, O'Neill A, Fan Y, Fitzpatrick JM, Dunn MJ, Watson RW J Proteome Res. 8:

34 HUMAN SALIVARY PROTEOME AND BITTER TASTE: THE RECIPROCAL INFLUENCE Tiziana Cabras, Melania Melis*, Alessandra Padiglia, Massimo Castagnola#, Irene Messana, Iole Tomassini Barbarossa* Department of Life Sciences and Environment, *and Department of Experimental Biology, Section of General Physiology, University of Cagliari, Monserrato, CA, Italy. #Institue of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy. Taste buds containing taste receptor cells are distributed mainly in the mucosa of the oral cavity, and owing the anatomical location they are closely associated with saliva secreted from the major and minor salivary glands. Thus, saliva represents a key element in the composition of the environment of the taste receptor cells, and changes in the quantity and quality of saliva may affect taste sensitivity. For instance, it has been already observed that proline-rich proteins (PRPs) and histatins can bind and precipitate in the oral cavity plant polyphenols (tannins) evoking the astringency sensation (Luck et al., 1994; Yan and Bennick, 1995). The purpose of this work was to analyze the possible relationship between bitter taste sensitivity and salivary proteome, and also to establish if and how the salivary proteome changes following a stimulation of taste receptors. Whole saliva of two groups of subjects with different sensitivity to bitter taste was analyzed by RP-HPLC-ESI-MS and the major salivary proteins quantified on the basis of the extracted ion current peak area. Only specific proteins belonging to the basic PRPs family showed a statistical significant different concentration in the two groups of subjects in basal conditions, and differences were even higher after stimulation. The present study evidences that specific salivary proteins are related to bitter taste. Corresponding author: Prof. Massimo Castagnola Institue of Biochemistry and Clinical Biochemistry Catholic University L.go F. Vito 1, Rome Italy Tel: Fax: [email protected] Yan Q., Bennick A. Identification of histatins as tannin-binding proteins in human saliva. Biochem J. 1995;311: Luck G., Liao H., Murray N.J., Grimmer H.R., Warminski E.E., Williamson M.P., Lilley T.H., Haslam E. Polyphenols, astringency and proline-rich proteins. Phytochemistry 1994;37: CAN PROTEOMIC APPROACH HELP US IN DIAGNOSIS OF RIEDEL S THYROIDITIS? Federica Ciregia a, Pietro Iacconi b, Laura Giusti a, Ylenia Da Valle a, Elena Donadio a, Gino Giannaccini a, Agnese Proietti b, Liborio Torregrossa b, Antonio Lucacchini a a Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa bdepartment of Surgery, University of Pisa The diagnosis of Riedel s thyroiditis, a rare thyroid disease, can be difficult to assess prior to surgical removal and can be confused with malignancy both clinically and cytologically. We present a case of a 71- year-old Italian woman who presented a goiter which showed a rapid increase in size at ultrasound check suggesting malignancy. Based on inconclusive cytology a total thyroidectomy was performed. Fine needle aspiration (FNA) of removed thyroid was processed by two dimensional electrophoresis and the proteome was compared both with anaplastic cancer and control samples. Protein pattern of Riedel s FNA revealed a superimposition with that of control. Previously, we found ferritin heavy chain, ferritin light chain and haptoglobin among the potential proteins biomarkers of thyroid malignancies1. So we performed western blot analysis of these proteins and we showed that their expression level were low or absent in Riedel s and control samples despite of high concentrations present in FNA anaplastic samples. These results suggest the potential applicability of FNA proteome analysis to concur to Riedel s diagnosis. 1Giusti et al., J Proteome Res ;7(9): Corresponding author: Dr Federica Ciregia, Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, Via Bonanno 6, Pisa, Italy, Tel: , Fax: , adress: [email protected]

35 2.10 COORDINATED CHANGES OF ADHESION ELEMENTS MAY REGULATE PHENOTYPE SWITCH IN VASCULAR SMOOTH MUSCLE CELLS Silvia Rocchiccioli1, Antonella Cecchettini1,2, Nadia Ucciferri1,2, Maria Giovanna Trivella1, Lorenzo Citti1 1 Istituto di Fisiologia Clinica-CNR, Pisa, Italy 2 Dipartimento di Morfologia umana e Biologia applicata, Università di Pisa, Pisa, Italy Introduction: Vascular smooth muscle cells (VSMCs) can switch from quiescent-contractile to migratory-synthetic states regulating the homeostasis of the vascular wall [1]. An imbalance in VSMC functions leads to vascular disease and their migration contributes to neo-intima formation after vascular injury in restenosis and atherosclerosis [2]. Methods: Protein extracts from quiescent and activated VSMCs were fractionated by anion-exchange extraction. Eluted fractions were selected and, after digestion, peptide mixtures were fractionated by HPLC and collected for MALDI TOF/TOF analysis. Differential protein expression was performed using spectral counts and confirmed in two cases with ELISA test. Immuno-fluorescence confocal microscopy was used to evidence differences in protein localization in the two phenotypes. Results: In the present work the anion-exchange extraction [3] allowed us to identify 360 proteins and of these, 20 were found markedly altered in activated VSMCs. The expression of two of them was validated by ELISA test. Immunofluorescence confocal microscopy was used to evidence colocalization of vinculin and talin-1 in activated phenotype, Conclusion: Eight differentially modulated proteins (Fibronectin, vimentin, vinculin, filamin A, LASP, talin-1, PDZ and LIM domain protein1, caldesmon) are directly involved in the formation and modulation of adhesion structures suggesting a pivotal role of these protein complexes in the phenotypic modulation. The implications of these proteins in cell migration are reported [4] but it is the first time that a simultaneous displacement and quantitative shift of a protein family is described in VSMCs. This study represents the first step to ascertain the actors of cell activation, their roles and interactions. Corresponding author: Lorenzo Citti Istituto di Fisiologia Clinica-CNR Via Moruzzi 1, Pisa (Italy) [email protected] Tel: Fax: [1] Chamley-Campbell, J., Campbell, G. R., Ross, R., The smooth muscle cell in culture. Physiol. Rev. 59, 1-6 [2] Owens, G. K, Kumar, M. S., Wamhoff, N. R., Molecular regulation of vascular smooth muscle cell differetiation in development and disease. Physiol. Rev. 84, [3] Rocchiccioli, S., Citti, L., Boccardi, C., Ucciferri, N., Tedeschi, L., Lande, C., Trivella, M. G., Cecchettini, A., A gel-free approach in vascular smooth muscle cell proteome: perspectives for a better insight into activation. Proteome Science 8, 15 [4] Manzanares, M. V., Choi, C. K., Horwitz, A. R., Integrins in cell migration-the actin connection. J. Cell Science 122, IDENTIFICATION OF NUCLEAR SUBSTRATES OF AKT/PKB BY FUNCTIONAL PROTEOMICS: PROHIBITIN 2 IS A TARGET OF AKT PHOSPHORYLATION IN HUMAN PROMYELOCYTIC LEUKEMIA CELLS. Antonietta D Angelo1,2, Alberto Bavelloni1,2, Manuela Piazzi3, William Blalock3, Francesca Tagliavini 3, Irene Faenza 3, Diego Pinetti 4, Lucio Cocco 3. 1RAMSES, Rizzoli Orthopedic Institute, Bologna, Italy 2Laboratory of Musculoskeletal Cell Biology, Rizzoli Orthopedic Institute, Bologna, Italy. 3Cellular Signaling Laboratory, Department of Human Anatomical Sciences, University of Bologna 4CIGS, University of Modena e Reggio Emilia, Italy The serine/threonine protein kinase Akt is a major signal transducer of the phosphoinositide 3-kinase (PI 3-K) pathway in all cells and tissues and plays a pivotal role in the maintenance of cellular processes including cell growth, proliferation, survival, metabolism and development of many malignancies including acute myeloid leukemia. The frequent aberrant activation of the PI 3-K/Akt pathway in human cancer has made it an attractive therapeutic target. Therefore, the study of effector proteins downstream of Akt could clarify the role of Akt in the development of myeloid leukemia. Although both localization and activity of Akt in the nuclear compartment are well documented, most Akt substrates identified so far are located in the cytoplasm, while nuclear substrates have remained elusive. In this study, we applied a proteomic approach to identify novel Akt substrates by using an antibody that recognized a consensus motif phosphorylated by Akt (K/RXK/RXXS/T) when phosphorylated on S/T (anti-phospho-akt substrate antibody). NB4 cells were treated with ATRA, and the putative Akt substrate proteins were isolated by immunoprecipitation with the anti-phospho-akt substrate antibody. The proteins were separated on SDS-PAGE and analyzed by ESI-Q-TOF mass spectrometry. This analysis indicated prohibitin 2, a potential tumor suppressor protein with potent transcriptional functions in the nucleus, as a putative substrate of Akt in the nucleus of NB4 cells. The putative Akt-Prohibitin 2 interaction was validated by reverse in vivo immunoprecipitation from nuclear protein of NB4 cells. In vitro phosphorylation of endogenous prohibitin 2 by recombinant Akt further validated this result. Dr. Antonietta D Angelo RAMSES, Rizzoli Orthopedic Institute Via di Barbiano, 1/ Bologna [email protected] Phone: Fax: References: Vandermoere F, El Yazidi-Belkoura I, Demont Y, Slomianny C, Antol J, Lemoine J, Hondermarck H. Proteomics exploration reveals that actin is a signaling target of the kinase Akt. Mol Cell Proteomics Jan;6(1): Kasashima K, Ohta E, Kagawa Y, Endo H. Mitochondrial functions and estrogen receptor-dependent nuclear translocation of pleiotropic human prohibitin 2. J Biol Chem Nov 24;281(47):

36 2.12 PROTEOMIC PROFILE OF WASHING FLUID OF COLORECTAL TRACT TO SEARCH POTENTIAL BIOMARKERS OF COLON CANCER Ylenia Da Valle a, Pietro Iacconi b, Federica Ciregia a, Laura Giusti a, Tiziana Ventroni a, Elena Donadio a, Gino Giannaccini a, Massimo Chiarugi b, Liborio Torregrossa b, Agnese Proietti b, Fulvio Basolo b, Antonio Lucacchini a a Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, Pisa, Italy b Department of Surgery, Pisa, Italy Washing fluid (WF) of colon rectal tract after surgical resection might represent a first step to obtain a mixture of proteins deriving from secretion of tumoral epithelial cells and then potentially involved in the pathological progression of tissue. In this study we performed a proteomic analysis of colorectal WF, to search potential biomarkers of colon cancer. The consequence of this approach might open the possibility to use the WF before surgery, to screen precancerous and early stage of colorectal cancer. Samples of WF were obtained at surgery from 42 patients submitted to colon resection for adenocarcinoma or carcinoma. Fifty millilitre of physiological solution were used to wash the internal side of resected tract. The WF were immediately centrifuged, concentrated (Millipore NMWL5000) and TCA added to obtain protein pellets. The samples were grouped in three classes: adenocarcinoma (AC), adenocarcinoma with lymph-node invasion (ACL) and ascending adenocarcinoma (ACA). After 2DE the protein patterns of malignant samples were compared with respective normal samples. A total of 83 protein spots were found differentially expressed exhibiting 2 fold-change of mean value spot intensities. After mass spectrometry these protein spots collapsed in 43 different proteins. Beside of alfa-1 antitrypsin and heat shock proteins, overexpressed in all groups, peculiar proteins resulted differentially expressed in ACL and ACA (i.e selenium binding protein, plastin-2, serpin B5, serpin B9, PCNA, translationally controlled tumour protein, elastase 3B). Our results suggest that WF could be a starting point to study the presence of potential markers implicated in tumour onset and progression. Dr Ylenia Da Valle Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology Via Bonanno Pisa Italy adress: [email protected] Tel: Fax: LIPIDOMIC APPROACH ON URINARY EXOSOMES FROM RCC PATIENTS: AN INSIGHT INTO THE TUMOR BIO-SIGNATURE Piero Del Boccio a,b,, Francesca Raimondo c, Damiana Pieragostino a,b, Lavinia Morosi c, Francesco Rocco d, Paolo Brambilla c, Claudia Augello e, Andrea Urbani b, Paolo Sacchetta a,b, Fulvio Magni c, Marina Pitto c a Dept. of Biomedical Science, University G. d Annunzio of Chieti-Pescara Italy b Research Center on Aging (Ce.S.I), University G. d Annunzio of Chieti-Pescara Italy cdept. of Experimental Medicine, University of Milano- Bicocca, Monza, Italy ddept. of Urology, Hospital Maggiore IRCCS, University of Milano, Italy edept. of Medicine Surgery and Dentistry, Pathological Anatomy, S. Paolo Hospital, Milano, Italy Urinary exosomes provide a non-invasive means of acquiring unique information about the physiological or pathophysiological state of their renal cells of origin. Exosomes are delivered to the urine from all renal epithelial cell types. Consequently, analysis of urinary exosomes may provide a source of biomarkers for diseases. Clear cell renal carcinomas (RCC) is representing about 3% of all kidney cancers. No biomarkers for early diagnosis of RCC are yet available. Here we addressed the lipidomic analysis of the exosomes isolated from control and RCC patient urines in order to look for specific lipid composition to be exploited as potential tumor signature. We demonstrated that CapLC Q-TOF MS was useful and helpful to characterize the lipidome of urinary exosomes showing good analytical performance for comparative analysis in terms of repeatability and mass accuracy. Differential lipid composition in RCC urinary exosomes was highlighted and tentatively identified by accurate mass and MS/MS experiments, giving evidence of a relationship between lipid composition of urinary exosomes and RCC disease. Our results show lipidome changes in RCC urinary exosomes opening the route for the discovery of novel potential tumour marker, for better understanding unusual membrane organization in pathological condition, for identifying bioactive molecules involved in signalling functions and, overall, for improving our knowledge of the molecular mechanisms involved in RCC onset and progression. Piero Del Boccio, Ph.D. Department of Biomedical Sciences "G. d'annunzio" University Via dei Vestini 31, Chieti Pescara, Italy [email protected] phone: Fax: Mobile:

37 2.14 ANALYSIS OF TRASTUZUMAB EFFECTS IN BREAST CANCER CELLS IN VITRO Gianluca Di Cara1,2, Nadia N. Albanese1,2, Rosa Musso1,2, Francesca Costantini1,2, Maria R. Marabeti1,2, Patrizia Cancemi1,2, Ida Pucci- Minafra1,2 1. Dipartimento Biomedico di Medicina Interna e Specialistica, Sezione di Oncologia Sperimentale e Applicazioni Cliniche, University of Palermo, Italy. 2. Centro di Oncobiologia Sperimentale (COBS), Palermo, Italy. Trastuzumab is a monoclonal antibody directed against the Her-2 oncoprotein extracellular domain. HER2 is a membrane receptor belonging to the human EGF receptors (EGFRs) subfamily together with HER-1, HER-3 and HER-4. All these receptors are involved in the normal development of the mammary gland, both at anatomical and physiological levels, but their aberrant expression occurs in a large number of breast carcinomas. HER-2 amplification/over-expression occurs in the 25/30% of breast cancer (BC) patients and is considered a predictive and prognostic marker for clinical approach to the evaluation of BC progression and a new therapeutic target for the treatment with Trastuzumab-based drug (Herceptin ). In the last two decades Trastuzumab treatment improved anti-cancer clinical approach by reducing the mortality among HER-2 overexpressing patients. Nevertheless the strong initial response to Trastuzumab based regimen is often followed by drug resistance occurring within 1 year in the majority of patients. To date several hypotheses were made about molecular bases of acquired Trastuzumab resistance, but the identification of molecular actors closely related to this phenomenon has not yet been fully performed. The aim of present study is the identification by proteomic strategy of protein clusters involved in early (sensitive cells, HS) and late response (resistant cells, HR) to trastuzumab in vitro, in order to select and evaluate predictive markers of resistance in vivo. SKBR-3 cells were utilized as a model for present study, because of their HER-2 gene amplification and protein strong over-expression. In order to evaluate Trastuzumab effects on the growth rate of cells, SKBR-3 were treated with 10µgr/ml of drug for 7 days: cell number was determined performing a MTS assay (based on tetrazolium salt degradation). To obtain sensitive (briefly treated) and resistant (long treated) clones we treated SKBR-3 cells respectively for 7 and 75 days (until cells growth rate became synchronous compared to untreated parental cells) with 10µgr/ml of drug. Parental, sensitive and resistant cells were then properly collected, lysed and subjected to 2D- IPG based proteomic analysis. Proteins identified by MALDI-TOF mass spectrometry were then clusterized by using DAVID gene ontology database online. Our results suggest that Trastuzumab treatment affects protein expression in a time-dependent manner with regard to proteins involved in glycolytic pathway, cytoskeleton remodeling, stress response, cell motility, calcium-dependent processes. We suggest that present data may improve significantly the knowledge of the mechanisms underlying trastuzumab drug resistance on BC cells, useful for clinical applications PROTEIN CARBONYLATION IN PERIPHERAL BLOOD MONONUCLEAR CELLS FROM AMYOTROPHIC LATERAL SCLEROSIS PATIENTS Assunta Gagliardi1, Simona Frosali2, Claudia Landi1, Laura Bianchi1, Maria Cipriano1, Michele Puglia1, Anna Gimigliano1, Alessandro Armini1, Anna Di Stefano2, Luca Bini1. 1Functional Proteomics Laboratory, Department of Biotechnologies, University of Siena, Siena, Italy. 2Department of Biotechnologies, University of Siena, Siena, Italy. Amyotrophic Lateral Sclerosis (ALS) is a progressive and fatal neurodegenerative disease characterized by selective death of motor neurons in the spinal cord, brain stem and motor cortex leading to muscular weakness and atrophy. The ALS pathogenesis is largely unknown and many studies agree on that it is a multifactorial disease. Oxidative stress is one of the mechanisms that have been hypothesized to be involved in the pathogenesis of ALS. Studies suggest the presence of oxidative stress markers in the motor cortex and brain stem, in cerebrospinal fluid and plasma from ALS patients. The most widely studied oxidative stress-induced protein modification is carbonylation. Our purpose was to characterize by a proteomic approach the carbonylated proteins of peripheral blood mononuclear cells (PBMCs) from ALS patients, in order to define potential protein markers of oxidative stress in ALS. PBMCs were isolated from whole blood obtained from age-matched ALS patients and healthy controls. Proteins were appropriately extracted and separated by high-resolution 2D-electrophoresis. Then, carbonylated proteins were detected by Immunoblotting using specific antibodies against carbonyl groups. Image analysis, performed with ImageMaster 2- D Platinum 7.0 software, allowed us to detect some proteins with qualitative and quantitative differences and frequency of carbonylation among ALS patients and healthy controls. In conclusion, the proteomic approach to the study of ALS could be useful to search for protein biomarkers that could provide insight into pathogenetic mechanisms of ALS and that might be important for the diagnosis and prognosis of the disease. Acknowledgment: this work was supported by the FIRB project Italian Human ProteomeNet (BRN07BMCT_013), from the MIUR. Dr. Assunta Gagliardi, PhD student University of Siena Department of Biotechnologies Functional Proteomics Laboratory Via Fiorentina, Siena, Italy [email protected] Phone: (+39) Fax: (+39) Gianluca Di Cara Post doc student Centro di Oncobiologia Sperimentale (C.OB.S.) Via San Lorenzo Colli 312/d [email protected] Tel: Fax:

38 2.17 A PROTEOMIC STUDY OF HEPATOCELLULAR CARCINOMA DISEASE: A COMPARISON BETWEEN THE LIVER INFLAMMATORY CONDITION AND CANCER OUTCOME BY DIGE APPROACH Gimigliano Anna a, Martins M.Aline b, Bianchi Laura a, Puglia Michele a,landi Claudia a Gagliardi Assunta a, Cipriano Maria a, Armini Alessandro a, Bini Luca a a Functional Proteomics Laboratory, Biotechnology Department, Siena University, Siena - Italy b National Experimental Oncology Laboratory, Physiology and Pharmacology Department - Brazil Hepatocellular carcinoma (HCC) is a widely increasing disease condition associated to heterogeneous factors, however the major risk determinant is liver chronic inflammation caused by hepatitis virus B (HBV), hepatitis virus C (HCV) or less frequently alcohol consumption. Until now the only potentially curative treatment of HCC remains the liver transplantation, even if for selected patients, so that mortality incidence is very high. The aim of this work is to understand the mechanism underlying the liver carcinogenesis developing from a chronic inflammatory environment. The quantitative 2D-DIGE (Two Dimensional-Differential In Gel Electrophoresis) method was selected to analyse human biopsy samples. Patients affected with chronic inflammation by HBV, HCV infection or alcohol abuse were compared to biopsies from patients characterized by HCC with corresponding pathological background: HBV-HCC, HCV-HCC, Alcohol-HCC. The collected samples from the inflammatory and HCC experimental conditions were alternatively labelled with Cy3 or Cy5 dyes and run along with a first and a second dimension. The resulting spot maps were analysed by the Decyder 2D Software v 7.0 (GE Healthcare). Many protein spots were visualized as differentially expressed in patients at the inflammatory step respect to the subjects with inflammation shifted to cancer. Mass spectrometry was performed to identify the putative implicated proteins. The proteomic approach could help to detect the variables involved in the molecular staging of HCC outcome, from inflammation to cancer lesions, in order to introduce new diagnostic tools for prevention and targeted therapies to control the tumour growth. This work was supported by the FIRB project Italian Human ProteomeNet (BRN07BMCT_013 - MIUR) to BL. Corresponding author: Anna Gimigliano [email protected] PhD, University of Siena Via Fiorentina 1, Phone: Fax: DIGE ANALYSIS OF OVARIAN CANCER CELL RESPONSES TO CYTOTOXIC GOLD COMPOUNDS AND ANTIMETASTATIC RUTHENIUM COMPOUNDS Francesca Guidi a,d, Michele Puglia b, Francesca Magherini d, Luca Bini b, Enrico Mini c, Chiara Gabbiani a, Luigi Messori a and Alessandra Modesti d a)department of Chemistry, University of Firenze, Sesto Fiorentino Firenze b)department of Molecular Biology, University of Siena Siena c)department of Preclinical and Clinical Pharmacology, University of Firenze Firenze d)department of Biochemistry, University of Firenze Firenze Platinum-based chemotherapy is the primary treatment for human ovarian cancer. Overcoming platinum resistance, using metal nonplatinum compounds (for example Au and Ru), has become a critical issue in current ovarian cancer chemotherapy, as drug resistance is the main reason for treatment failure1. Very little is known concerning molecular mechanisms underlying pharmacological effects of gold- and ruthenium-based antitumor metallodrugs. Notably, gold-compounds seem to exert their cytotoxic effects through mechanisms distinct from those of the classical anticancer platinum-complexes2. Ruthenium-compounds show important antimetastatic properties in vivo. We focused our attention on the effect of AuL12 and Au2Phen (gold) and NAMI-A and RAPTA-T (ruthenium) on A2780 human ovarian cancer cells (cis-platin sensitive). A classic 2D-DIGE proteomic approach is exploited here to gain deeper insight into those mechanisms3. First, cytotoxicity profiles of AuL12, Au2Phen, NAMI-A and RAPTA-T were determined against A2780 cells. Protein extraction from gold- and ruthenium-treated A2780 cells and 2D-DIGE separation were carried out according to established procedures. DeCyder software analysis was performed and differentially expressed proteins were visualized in these cases: 1) single compound vs control, 2) AuL12 and Au2Phen vs control, 3) NAMI-A and RAPTA-T vs control, 4) all compounds vs control. According to MALDI-TOF MS and ESI-Ion trap MS/MS several differentially expressed proteins were identified. We report here the preliminary results we obtained. These metallodrugs caused relatively modest changes in protein expression in treated cells compared to controls. In particular, four altered proteins were in common between all treatments. Among them we found overexpressed the protein Omega amidase NIT2, that is studied as a tumoursuppressor able to arrest cells in the G2 phase of the cell cycle. This work was supported by the FIRB project Italian Human ProteomeNet (BRN07BMCT_013), from Italian Ministry of University and Scientific Research and by Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici (C.I.R.C.M.S.B). Dr Francesca Guidi, PhD Dipartimento di Scienze Biochimiche Università degli studi di Firenze Viale Morgagni, 50-FIRENZE [email protected] Tel: Gallori E. et al.. Arch. Biochem. Biophys. (2000) 376: Casini A et al.. J Inorg Biochem. (2008) 102: Friedman DB, Lilley KS, Methods Mol Biol (2008). 428:93-124

39 2.19 PROTEOMIC ANALYSIS OF INTERSTITIAL LUNG DISEASES Landi Claudia a, Bargagli Elena a, Perari Maria Grazia a, Puglia Michele b, Gagliardi Assunta b, Cipriano Maria b, Bianchi Laura b, Gimigliano Anna b, Armini Alessandro b, Rottoli Paola a, Bini Luca b. a Department of Clinical Medicine and Immunological Science, section of pulmonary disease, University of Siena, Siena, Italy. b Functional proteomics Lab., Department of Biotechnology, University of Siena, Siena, Italy. The difficulty to distinguish, in terms of diagnosis and prognosis, different interstitial lung diseases (ILD) such as Idiopatic Pulmonary Fibrosis (IPF), Sarcoidosis (Sar), Pulmonary Langerhans Cell Histiocytosis (PLCH) and Pulmonary Fibrosis associated to Systemic Sclerosis (Ssc), is a major task in clinical studies. Proteomics could be a valuable methodological procedure to highlight specific biomarkers to characterize these diseases. Therefore we performed this study comparing Bronchoalveolar lavage (BAL) samples from patients with all these conditions and smoker (sc) and no-smoker (nsc) healthy controls. BAL samples, were resolved by 2D-Electrophoresis. Gel images were analyzed by Image Master Patinum 7.0 software. A statistical analysis was performed by a T-test. Multivariate statistical analysis, including Principal Component Analysis (PCA) to examine global trends in protein expression. MALDI-ToF/MS and LC-MS/MS were used to identified the differentially expressed proteins. Each condition was compared to the others to evidence the differentially protein pattern associated with a specific condition. A total of 339 proteins were found differentially expressed among the different diseases. Interestingly, some of these proteins were differentially expressed in more than one group, but some others were specific for one condition. Using a PCA on the differentially expressed proteins, the different gels of the analysis were grouped correctly in six groups, respectively IPF, Sar, PLCH, Ssc, sc, nsc. In conclusion proteomic analysis on ILD can help to identify proteins of interest to be analyzed as potential biomarkers to distinguish these pathologies and PCA can help to classify unknown samples in the appropriate group. This work was supported by the FIRB project Italian Human ProteomeNet (BRN07BMCT_013-MIUR). Dr. Claudia Landi, PhD student University of Siena Department of Clinical Medicine and Immunological Science Santa Maria Alle Scotte Hospital Viale Bracci, Siena, Italy [email protected] Phone: (+39) Fax: (+39) PROTEOMIC ANALYSIS OF HUMAN GLIOBLASTOMA CELL LINES WITH DIFFERENT RESPONSE TO NITRIC OXIDE. Roberta Leone A, Paola Giussani B, Agnese Viganò A, Michele Vasso A,C, Daniele CapitanioA, Chiara Fania A, Paola Viani B, Cecilia Gelfi A,C A Dipartimento di Scienze e Tecnologie Biomediche, Università degli studi di Milano, Segrate (MI), Italy B Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Università degli studi di Milano, Segrate (MI), Italy C Istituto di Bioimmagini e Fisiologia Molecolare, Consiglio Nazionale delle Ricerche, Segrate (MI), Italy The glioblastoma multiforme is the most aggressive astrocytoma tumor, affecting brain glial cells. Affected patients, even after aggressive treatments (e.g. surgical resection, radiotherapy and chemotherapy) have a median survival of less than one year. Nitric oxide (NO) has emerged as a potential anti-oncogenic agent able to overcome tumor resistance, unfortunately it isn t in all tumor cases. The development of cellular models resistant to various cytotoxic stimuli is crucial for the comprehension of molecular mechanisms at the basis of this phenomena. CCF-STTG1 and T98G cell lines, both derived from glioblastoma multiforme were adopted to monitor the effect of NO donors. CCF-STTG1 cells respond to treatment with different NO donors inhibiting proliferation with a ceramide-dependent mechanism, whereas T98G are totally resistant. Molecular differences between these two cell lines were assessed applying 2D-DIGE and mass spectrometry. Results indicated an increased abundance of chaperone and stress response proteins in T98G cell line. These changes can create an adverse environment for NO action, justifying T98G enhanced resistance. Furthemore, some proteins, involved in the pathway that regulates ceramide trafficking between ER and Golgi, resulted deregulated in T98G compared to CCF-STTG1, this probably counteracts ceramide accumulation in ER avoiding proliferation arrest. In conclusion, proteomic analysis highlighted a more effective control of the intracellular redox status and a more efficient ceramide removal from ER in T98G cell line, that can account for the intrinsic resistance to NO action. This protein set could provide new insight in the prediction of NO response in tumor treatment. Cecilia Gelfi Professor Department of Sciences and Biomedical Technologies - University of Milan Via F.lli Cervi 93, Segrate (MI), Italy [email protected] Phone: Fax:

40 2.21 MALDI IMAGING: A NEW TOOL FOR MAPPING MULTIPLE SCLEROSIS BRAIN LESIONS TO LOOK BEYOND CLASSICAL HISTOLOGY Giuseppina Maccarrone a*, Sandra Nischwitz a*, Søren-Oliver Deininger b, Joachim Hornung a, Fatima König c, Christine Stadelmann-Nessler c, Frank Weber a*, Chris W. Turck a* a Max Planck Institute of Psychiatry, Munich, Germany b Bruker Daltonics GmbH, Bremen, Germany c Department of Neuropathology, Georg-August- Universität, Göttingen, Germany * Both authors contributed equally Remyelination of multiple sclerosis (MS) brain lesions is an important mechanism to prevent chronic disability. However, the exact mechanism of remyelination remains elusive. Recently, neuropathological protein analysis using Mass Spectrometry Imaging has become feasible. This technique visualises protein and peptide spatial distributions and generates molecular maps within tissue sections. We employed Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI-MSI) to profile and to identify peptides and proteins expressed in normal-appearing white matter, grey matter and MS brain lesions with different extents of remyelination. Statistical analyses of mass spectra obtained from unsupervised hierarchical clustering and principle component analysis, generated molecular images. The spatial mass distributions were used to define regions within the tissue slide that agreed with those obtained from histological analysis. Areas, which showed little remyelination, were defined by low molecular compounds with mass-to-charge ratios (m/z) in the range from m/z 3000 to In more extensively remyelinated areas, compounds with high molecular weights in the mass range m/z to were detected. An indepth interrogation of the hierarchical clustered mass spectrometry dataset led us to the discovery of cortical lesions that were not visible by routine histology, but confirmed by subsequent Proteolipid Protein (PLP) and 2,3 -Cyclic Nucleotide 3 -Phosphodiesterase (CNPase) immunostainings. We can conclude that MALDI-MSI has the ability to distinguish lesions with different extents of remyelination, to detect cortical lesions that were not visible on routine histopathological stains and to define structurally distinct sub-regions of lesions with similar expression profiles and localisations of potentially disease-related proteins and peptides. Giuseppina Maccarrone, Dr. Max Planck Institute of Psychiatry Research group: Proteomics and Biomarkers Kraepelinstr.2-10, Munich, Germany [email protected] Tel: Fax: TIME-COURSE INVESTIGATION OF SAGM-STORED ERYTHROCYTE CONCENTRATES: FROM METABOLISM TO PROTEOMICS Cristina Marrocco, Gian M. D Amici, Angelo D Alessandro, Valeria Pallotta, Sara Rinalducci and Lello Zolla Department of Environmental Sciences, University of Tuscia, Viterbo, Italy In this study we performed an integrated biochemical, metabolomics and proteomics time-course investigation on SAGM-stored RBCs. We could confirm early accumulation of biochemical changes including ph drop, accumulation of potassium in the supernatant, while glycolytic metabolites levels increased over the first two weeks of storage. From day14 onwards, we could observe significant consumption of all metabolite species, and divertion towards the pentose phosphate pathway. These phenomena coincided with the accumulation of structural alterations. While at day14 RBCs seemed still viable, at day21 SEM images revealed that 50% of the RBCs displayed either reversible or irreversible shape profiles. Altered membrane shapes resulted in anomalous ESR and increased osmotic fragility. A wide-series of parameters which are routinely altered in hereditary spherocytosis patients, viz membrane structural protein fragmentation (spectrin, band3, band4.1), membrane accumulation of hemoglobin, antioxidant enzymes (peroxiredoxin-2) and chaperones, and the increase in oxidation markers (protein carbonylation and MDA accumulation) were all evidenced in ex vivo RBCs. We could thus correlate these observations to the onset of the vesiculation phenomenon, through a proteomics snapshot of the differential membrane proteome at day0 versus day35. We could detect proteins involved in vesicle formation and docking to the membrane, such as SNAP alpha. Lello Zolla Professor Tuscia University Largo dell Università snc, Viterbo, Italy address: [email protected] Tel.: Fax:

41 2.24 PROTEOMIC AND MORPHOLOGICAL CHARACTERIZATION OF RCC URINARY EXOSOMES Lavinia Morosi a, Francesca Raimondo a, Pamela Della Mina b, Antonello Villa b, Clizia Chinello a, G Cozzi c, Paolo Brambilla e, Fulvio Magni a and Marina Pitto.a a Department of Experimental Medicine, Univ. of Milano- Bicocca, Monza, Italy b Microscopy and Image Analysis Consortium, University of Milano-Bicocca, Monza, Italy c Hospital Maggiore IRCCS, Dept. of Urology, Univ. Milano, Italy d Pathology Unit, Dept. of Medicine, Surgery and Dental Sciences, Hospital of S. Paolo and IRCCS-Policlinico Foundation, Mangiagalli and Regina Elena, Univ. Milano, Italy e Department of Laboratory Medicine, Hospital of Desio, Monza, Italy Renal cell carcinoma (RCC) is among the 10 leading causes of cancerrelated deaths worldwide, and its incidence is increasing. Therefore, it is important to identify differentially expressed proteins to be used as potential RCC markers. A promising strategy is comparative proteomics of urinary exosomes (UE), membranous vesicles released by every epithelial cell type facing the urinary space(1). About 30 ml of urines were collected from 29 RCC patients and 28 healthy matched controls. Exosomes were then isolated by urine ultracentrifugation after cells and debris clearing(2). UE morphology, shape and dimension were examined by electron microscopy and morphometrical analysis. For protein MS identification, representative UE derived from 9 RCC and 9 CTRL urines were pooled and proteins separated by 4-12% gel electrophoresis. After Coomassie Blue staining, gels were cut in 45 contiguous slices and analyzed by LC-MS/MS after trypsin digestion. Results show that exosomes have spherical shape and diameter comprised between 30 and 50 nm, as reported(2), with no significant differences between RCC and CTRL UE. We report identification of 187 proteins from RCC patient UE, and 261 from CTRL UE. About 30% of the identified proteins result membrane associated while another 30% in RCC exosomes (only 15% in CTRL ones) are secreted. We assessed the presence of many typical exosomal proteins(3), such as TSG101, component of the ESCRT machinery, annexins, involved in trafficking and membrane fusion, and tetraspannins such as CD9. Moreover, proteins related to cellular adhesion and motility (i.e. Ezrin), communication (G-proteins), immunity and transport (i.e. CLIC1) were found. Name: Lavinia Morosi Title: PHD student Institution: Università degli Studi di Milano-Bicocca Address: via Cadore Monza [email protected] Phone: Fax: References: (1) Hoorn EJ et al., Nephrology; 2005; 10: (2) Pisitkun T et al., PNAS 2006; 101: (3) Mathivanan S. et al., Mol. Cell. Proteomics (2010) ; 9: Grants from FIRB: Rete Nazionale per lo studio del proteome umano (n. RBRN07BMCT) 2.25 DIFFERENTIAL PROTEOMIC ANALYSIS OF THYROID CARCINOMA CELL LINES Rosa Musso1,2, Gianluca Di Cara1,2, Nadia N. Albanese1,2, Francesca Costantini1,2, Alessia Pivetti1,2, Maria R. Marabeti1,2, Giovanni Zito2,3, Patrizia Cancemi1,2, Carla Giordano1,2 and Ida Pucci-Minafra1,2 1. Dipartimento Biomedico di Medicina Interna e Specialistica (Di.Bi.M.I.S.), Sezione di Oncologia Sperimentale e Applicazioni Cliniche, University of Palermo, Italy. 2. Centro di Oncobiologia Sperimentale (COBS), Palermo, Italy. Thyroid carcinomas are the 1-2% of all malignant tumors and, after those of the gonads, are the most common tumors of the endocrine system. Papillary thyroid carcinomas (PTCs) represent approximately 80% of all thyroid malignancies and are characterized by a good prognosis. Differently, anaplastic thyroid carcinomas (ATCs), which account for less than 5% of all thyroid cancers, is the most malignant thyroid neoplasm and is almost invariably fatal. Careful histopathological examination of ATC reveals that many of them contain a papillary structure or follicular components in focal areas. It is believed that ATC is derived from the follicular epithelial cells and represents a terminal dedifferentiation of preexisting differentiated carcinoma. Many genetic alterations have been implicated in thyroid carcinomas and involve mostly the aberrant activation of the MAP kinase pathway. Several studies suggested that BRAF (V600E) mutation (90% of all BRAF mutations) plays an important role in the early steps of the papillary carcinogenesis, following experimental observations on microcarcinomas. BRAF(V600E) is often found in ATC histotype, suggesting that this mutation may be involved in thyroid cancer progression to poorly differentiated and aggressive phenotypes. It seems that ATC is derived from a process of dedifferentiation of papillary phenotype, for alterations in oncogenes and/ or tumor suppressors; recently with the spread of theories about the involvement of stem cells in tumors it is assumed that the ATC derived from fetal thyroid cells. In this work we performed a differential proteomic analysis of two thyroid cancer cell lines: B-CPAP cell line, derived from PTC, and 8505C cell line, derived from ATC (both cell lines are characterized by the presence of BRAF (V600E) mutation in homozygosis). These cell lines represent important model for the study of in vitro differentiated and undifferentiated tumors. B-CPAP and 8505C cells were then properly collected, lysed and subjected to 2D-IPG based proteomic analysis. Proteins identified by Gel-Matching and MALDI-TOF mass spectrometry were then clusterized by using DAVID gene ontology database online. Our preliminary results show that the differentially expressed proteins are involved in the glycolysis, regulation of apoptosis and cell motility, reflecting the different phenotypic characteristics of cell lines. We suggest that our data may be useful for identifying potential molecular markers associated with the PTC and ATC, to be used in clinical applications, and consequently this study could significantly increase the knowledge of the mechanisms underlying the process of dedifferentiation. 3 Dr. Zito present adress: Genetics Department, Yale University School of Medicine, New Haven, CT, USA. Rosa Musso PhD student Sezione di Oncologia Sperimentale e Applicazioni Cliniche, Di.Bi.M.I.S. Via San Lorenzo Colli 312/d [email protected] Tel: ; Fax:

42 2.27 HEART PROTEOMICS OF GUINEA PIGS Gianluca Paredi1, Luca Ronda1, Stefano Bruno1, Simona Bertoni2, Elisabetta Barocelli2, and Andrea Mozzarelli1 1Department of Biochemistry and Molecular Biology University of Parma, Parma, Italy. 2Department of Pharmacological, Biological and Chemical Applied Sciences, University of Parma, Parma, Italy. The therapeutic class of hemoglobin-based oxygen carriers (HBOCs)1 aims to substitute/complement the transfusion therapy in hemorrhagic shock or in anemic states. A meta analysis highlighted that the administration of hemoglobin-based oxygen carriers causes a significantly increased risk of death and myocardial infarction, that are not correlated with a particular class of HBOCs or a particular clinical practice2. Moreover, some of the same adverse effects arise in diseases associated with transfusions with aged blood and in hemolytic pathologies. The aim of our research is to develop a biochemical platform for total body safety assessment based on organ proteomics and standard blood clinical assays in order to identify the mechanisms involved in the adverse effects caused by hemoglobin-based oxygen carriers. In vivo experiments were performed in guinea pigs because their plasma redox potential is similar to that of humans. A 50% hemoglobin dilution was carried out by withdrawing half of the blood volume from 5 anaesthetized guinea pigs and replacing it with a blood expander. Proteomic analysis was focused on heart that is the organ mainly associated to the observed adverse effects. The heart proteins separation was obtained by gel electrophoresis (2D-PAGE and 1D-PAGE). An average of 138 protein spots were identified in gel by silver staining. A visual inspection of the gels revealed that most of the proteins are located in the ph range 5-8. The most abundant spots were identified by MALDI- TOF and LTQ-Orbitrap mass spectrometry analysis. Ongoing proteomic analysis is aimed to characterize subcellular compartments. Gianluca Paredi Department of Biochemistry and Molecular Biology University of Parma Via GP Usberti 23/A Parma, Italy [email protected] Phone: Fax: [1] Bruno S, Ronda L, Faggiano S, Bettati S, Mozzarelli A (2010). Oxygen delivery via allosteric effectors of haemoglobin and blood substitutes. Burger s Medicinal Chemistry, Drug Discovery and Development, 7th Ed [2] Natanson et al. Cell-Free Hemoglobin-Based blood substitutes and risk of myocardial infarction and death, JAMA 2008, 299,

43 2.28 DOPAMINE RECEPTOR AGONISTS AND LEVODOPA MODULATE PROTEIN LEVELS IN HUMAN T LYMPHOCYTES. Agnese C. Pippione a, Tiziana Alberio a, Daniela Cecconi b, Simone Olgiati a, Maurizio Zibetti c, Leonardo Lopiano c, Mauro Fasano a. a Department of Structural and Functional Biology and Centre of Neuroscience, University of Insubria, Busto Arsizio (VA) b Department of Biotechnology, University of Verona, Verona c Department of Neuroscience, University of Torino, Torino. Dopamine receptor agonists and Levodopa are used to treat symptoms in patients affected by Parkinson s Disease (PD) to counteract dopamine (DA) loss at the striatum level1. (T)-Lymphocytes express some features of the dopaminergic system (they express tyrosine hydroxylase, dopamine receptors and transporters, and store catecholamines into vesicles2), so their functions or activation can be regulated by dopamine. Several effects of dopamine on immune cells have been described3,4, but none of these studies focus on proteome modifications of human T-cells. In the attempt to establish if T-cells undergo modifications at the protein level after administration of DA agonists or Levodopa in human subjects, we enrolled 9 PD patients under DA agonist therapy and 5 PD patients DA agonist free. We compared two-dimensional electrophoresis maps of total proteins from T-cells and we identified 7 proteins whose level was significantly different in the two groups considered. Eleven among these patients were treated with Levodopa; indeed, we found levels of 4 spots correlating with the dose of Levodopa assumed daily by patients. These findings demonstrate that DA stimulation (either by Levodopa or DA agonists) has important effects on T-cell proteome in patients under long term treatment. Therefore, therapies acting on the dopaminergic system have additional effects on the immune system that cannot be neglected. In this view, studies that assert alterations in lymphocytes of PD patients have to take into account the therapy administered to patients. Corresponding author: Agnese Chiara Pippione PhD student Department of Structural and Functional Biology and Centre of Neuroscience, Laboratory of Biochemistry and functional Proteomics, University of Insubria Via A. Da Giussano 12, Busto Arsizio (VA), Italy [email protected] Phone: Fax: References: 1. Bonuccelli et al., Parkinsonism Relat. Disord. 2009; 15, S44-S Pacheco et al., J. Neuroimmunol. 2009; 216, Sarkar et al., Brain Behav. Immun. 2010; 24, Alberio et al., Biochimie 2011; 93, PROTEOMICS OF RCC MEMBRANE MICRODOMAINS Raimondo Francesca a, Morosi Lavinia a, Mainini Veronica a, Bianchi Cristina a. Albo Giancarlo b, Ferrero Stefano c, Magni Fulvio a and Pitto Marina a a Department of Experimental Medicine, Univ. of Milano- Bicocca, Monza, Italy b Hospital Maggiore IRCCS, Dept. of Urology, Univ. Milano, Italy c Pathology Unit, Dept. of Medicine, Surgery and Dental Sciences, Hospital of S. Paolo and IRCCS-Policlinico Foundation, Mangiagalli and Regina Elena, Univ. Milano, Italy Clear cell renal carcinomas (ccrcc) is representing about 3% of all kidney cancers. No biomarkers for early diagnosis of ccrcc in asymptomatic patients or for post-surgery monitoring are yet available. A promising strategy is subcellular comparative proteomics, allowing to enrich specific cell compartments and assess differences in protein expression patterns. We investigated the proteomic profile of a peculiar subcellular compartment, plasma membrane microdomains (MD), involved in cell signalling, transport, proliferation and in many human diseases, such as cancer. Subcellular fractions were prepared by differential centrifugation from surgical samples of RCC and adjacent normal kidney (ANK). MD were isolated from plasma-membrane-enriched fractions after Triton X-100 treatment and sucrose density gradient ultracentrifugation. MD derived from RCC and ANK tissues of 7 patients were pooled, and proteins separated by 4-12% and 12% gel electrophoresis. After Coomassie Blue staining, bands were excised and analyzed by LC-MS/MS after trypsin digestion. We identified 83 proteins from MD isolated from RCC tissue, and 95 proteins from ANK. About 60% of the identified proteins are membraneassociated and about half of these resulted microdomain-associated. GRAVY scores assignment shows that most identified proteins (about 70%) are in the hydrophobic range. From a functional point of view, we found proteins involved in signal transduction (Ras related proteins), channels (Aquaporin-1), carriers (Pglycoprotein) and cytoskeleton structural constituents (Spectrin). We chose a panel of proteins to validate their differential expression by WB. In conclusion, the recognition of differential amounts of plasma membrane proteins in RCC microdomains looks promising in view of new biomarker discovery. Name: Marina Pitto Title: Professor Institution: Università degli Studi di Milano-Bicocca Address: via Cadore Monza [email protected] Phone: Fax: Grants from FIRB: Rete Nazionale per lo studio del proteome umano (n. RBRN07BMCT)

44 2.30 CREUTZFELDT-JAKOB DISEASE AND OTHER DEMENTIA MAY BE DISCRIMINATED BY THYMOSIN BETA 4 LEVELS IN CEREBROSPINAL FLUID USING MALDI-TOF MS. Elena Urso1, Maria Le Pera1,, Sabrina Bossio2, Teresa Sprovieri1, Ida Manna1, Chiara Cupidi3, Umberto Aguglia4, Antonio Gambardella1, Aldo Quattrone5, Antonio Qualtieri1. 1) Institute of Neurological Sciences, National Research Council, Mangone, Cosenza, Italy 2) U.O.C. di Ematologia, Azienda Ospedaliera Annunziata, Cosenza, Italy 3) Department of Clinical Neurosciences. University of Palermo 4) Neurologic Unit, Azienda Ospedaliera Bianchi- Melacrino-Morelli, Reggio Calabria, Italy 5) Neuroimaging Research Unit, Institute of Neurological Sciences, National Research Council, Catanzaro, Italy Objective: to evaluate the diagnostic specificity and sensitivity of differentially expressed levels of Thymosin beta 4 (TB4) in cerebrospinal fluid (CSF) from Creutzfeldt-Jakob disease (CJD) patients and other dementia, using a mass spectrometry (MS)-based method. Methods: CSFs were obtained from 25 normal controls, 15 patients with CJD (4 genetic, 11 sporadic), 18 patients with Alzheimer s disease (AD), 16 patients with frontotemporal dementia (FTD) and five patients with limbic encephalitis (LE). Samples were subjected to desalting by reverse phase micro-chromatography (Zip-Tip) then to matrix assisted laser desorption ionization time of flight (MALDI-TOF) MS protein profiling analysis. Ions with a signal equal or superior to 1% were collected. The signal intensities were normalized presenting the values as area percent of the total area of all the signals. Significances ( p) were tested using the Student s t-test. A p value < 0.02 was considered significant. Results: The relative peak area (area percent) of TB4 appeared to be significantly increased in CJD samples compared to controls (p<10-9) and to all other groups. No significance was observed between AD (p=0.052), FTD (p=0.091) versus controls while we observed a low statistical significance for LE (p= 0.013) versus controls. At a cut-off value of 1.2 % of the TB4 peak area between CJD and the FTD+AD+LE group we have estimated a 100% of sensitivity and a 97.4% of specificity. Discussion. Recently applying a proteomic analysis on CSF from CJD and normal controls, we have been able to identify TB4 as a new candidate diagnostic biomarker for CJD. In the present work in order to validate the previously results, we report finding concerning TB4 levels in CSF from AD, FTD and LE. Our data clearly suggest that quantification of TB4 in CSF is an useful test in supporting antemortem diagnosis of CJD showing excellent sensitivity and specificity equal or superior to protein, that currently is included in the diagnostic criteria for CJD. There are not definitive data concerning the expression of TB4 in the central nervous system but recent works have shown its overexpression in glial cells. The elevated levels detected by us in CJD might reflect than the well known glial reaction found in the brain of the CJD patients. Conclusions: our results let us to propose TB4 as a new validate candidate marker to discriminate Creutzfeldt-Jakob disease from other dementia and encephalitis PROTEOMIC PROFILE OF CANCER STEM CELLS DIFFERENTIATION SHOWS ALTERATION OF GLYCOLYTIC PATHWAY Domenico Ciavardelli a, b*, Claudia Rossi b, c*, Federica Forlì b,c, Daniela Barcaroli b,c, Mirco Zucchelli b, c, Simona D Aguanno d, e, Paolo Sacchetta c, Carmine Di Ilio b, c, Matilde Todaro f, Giorgio Stassi f, Vincenzo De Laurenzi b,c and Andrea Urbani d, e a Faculty of Motor and Health Science, Kore University, Enna Italy. b Center of Excellence on Aging (Ce.S.I.), Chieti, Italy. c Department of Biomedical Science, University G. d Annunzio, Chieti-Pescara, Italy. c Department of Biology, University of Rome Tor Vergata, Italy. d University of Rome Tor Vergata, Department of Internal Medicine, Rome Italy. e IRCCS S. Lucia, Rome, Italy. f Department of Surgical and Oncological Sciences, University of Palermo, Italy. Cancer stem cells (CSCs) have a key role in solid tumours development, recurrence, and resistance to pharmacological treatment. In vitro, CSCs grow as three-dimensional cellular aggregates, called spheroids. Breast CSCs have been found to be resistant to conventional chemotherapy in vitro and in animal models and are thought to be able to regenerate the tumor after the bulk of more differentiated cells have been killed by therapy. To further characterize CSCs features we proposed a label free shotgun proteomics analysis to compare the protein expression profiles in CSCs grown as spheres or allowed to differentiate for 3.5 and 7 days. Our results show that the majority of changes occur already after 3.5 days of differentiation, indeed we identified 44 proteins down-regulated at this time point as compared to spheres. 19 of these were still down-regulated after 7 days of differentiation and only the expression of 4 additional proteins was reduced only after 7 days. Interestingly only 10 proteins increased in differentiated cells as compared to spheres. Functional analysis of the differentially expressed proteins reveals that CSCs have higher expression of a number of proteins related to carbohydrate metabolism and glycolysis, and of proteins related to free radical scavenging. Our findings suggest that breast CSCs exhibit a phenotype characterized by a higher glucose metabolism and increased defence from oxidative stress. Further studies are required but it is intriguing to speculate that breast CSCs preferentially perform anaerobic glucose metabolism on the basis of the significant increase of LDHA and PKM2 expression levels. *These authors equally contributed to the study. Corresponding author: Claudia Rossi, PhD Center of Excellence on Aging (Ce.S.I.) University G. d Annunzio of Chieti Via Colle dell Ara Chieti, Italy [email protected] Phone: Fax:

45 2.32 VALIDATION OF A BIOMARKER PANEL FOR RCC Claudia Salemi a,b, Daniela Fumagalli a,b, Francesca Raimondo a, Clizia Chinello a, Francesco Rocco c, Stefano Ferrero d, Cecilia Sarto b, Marina Pitto a, Paolo Brambilla a,b and Fulvio Magni a,b. a Department of Experimental Medicine, Univ. of Milano- Bicocca, Monza, Italy b Department of Laboratory Medicine, Desio Hospital, Monza, Italy c Hospital Maggiore IRCCS, Dept. of Urology, Univ. Milano, Italy d Pathology Unit, Dept. of Medicine, Surgery and Dental Sciences, Hospital of S. Paolo and IRCCS-Policlinico Foundation, Mangiagalli and Regina Elena, Univ. Milano, Italy The timely diagnosis and therapeutic monitoring of human renal cell carcinoma (RCC) is limited by lack of specific biomarkers; in fact to date, stage and tumor grade are the main prognostic indicators. Therefore, the search for new markers is of great importance. The aim of this work was to validate the differential expression in RCC of a protein panel including: ANXA2, LGAL1, CypA [1] and FABP7 [2]. To identify candidate biomarkers, we used Two-Dimensional Difference Gel Electrophoresis (2-D DIGE), combined with mass spectrometry (MS) for protein identification, on 24 matched sample of RCC tumor tissues and normal cortex (ANK, Adjacent Normal Kidney). Validation was performed by 1-D and 2-D Western blotting with specific antibodies. The results show a significant over-expression of the 4 proteins in tumor tissues and are in agreement with 2D-DIGE experiments. Moreover, the presence of different isoforms of ANXA2, LGAL1 and CypA in RCC suggests the presence of different post translational modifications, such as phosphorylation, which are currently under investigation [3,4]. The presence of ANXA2 in the urine of patients and healthy donors was also investigated in order to determine whether the differential expression observed in the kidney tissue could be also detected in this easily available biological fluid. Preliminary results show higher protein levels in the urine of healthy subjects compared to RCC patients, suggesting a putative role of ANXA2 in tumor progression. Our results could constitute the basis for the development of a multiple biomarker panel to be used for RCC prognostics and diagnostics. Claudia Salemi, doctor, Department of Laboratory Medicine, Desio Hospital, Monza, Italy. [email protected]. Te l References: [1] Lee J. Role of Cyclophilin A during Oncogenesis. Arch. Pharm. Res., 2010; 33 (2): [2] Domoto T., Miyama Y., Suzuki H., Teratani T., Arai K., Sugiyama T., Takayama T., Mugiya S., Ozono S., and Nozawa R. Evaluation of S100A10, annexin II and B-FABP expression as markers for renal cell carcinoma. Cancer Science, 2007; 98: [3] Deora A. B., Kreitzer G., Jacovina A. T. and Hajjarr K. A. An Annexin 2 phosphorylation Switch Mediates p11-dependent Translocation of Annexin2 to the Cell Surface. The Journal of Biological Chemistry, 2004; 279: [4] Eustace A. J., Dowling P., Henry M., Doolan P., Meleady P., Clynes M., Crown J., O Donovan N. 2-D DIGE analysis of phospho-enriched fractions from dasatinib-treated melanoma cell lines. Journal of Proteomics, 2011, 74: Grants from FIRB: Rete Nazionale per lo studio del proteoma umano (n. RBRN07BMCT) LYMPHOBLASTOID PROTEOME STUDY IN BIPOLAR DISORDER PATIENTS TREATED WITH LITIUM. Alessio Soggiu*1, Cristian Piras2, Alessio Squassina3, Maria Del Zompo3,4 Luigi Bonizzi5, Paola Roncada6 1 Section of Neurophysiology and Neurochemistry, Department of Neurosciences B.B. Brodie, University of Cagliari, (Cagliari), Italy 2 Department of Zootechnical Science, University of Sassari, Italy 3 Section of Clinical Pharmacology, Department of Neurosciences B.B. Brodie, University of Cagliari, (Cagliari), Italy 4 Unit of Clinical Pharmacology of the University Hospital of Cagliari, Italy 5 DIPAV, Facoltà di Medicina Veterinaria, Università degli Studi di Milano, 6 Istituto Sperimentale L. Spallanzani, Milano, Italy. Suicide is strongly associated with Major Affective Disorders with a risk for completed suicide 6 to 15 times higher in patients compared to the general population. Lithium is to date the most effective treatment for bipolar disorder (BD) with a well established protective effect against suicide. However, the mechanisms responsible for these clinical effects remain unclear. So far, several studies attempted to identify biological markers [1, 2] and genes[3] involved in suicide. To extend these findings, lymphoblastoid cell lines (LCLs) (n = 48) of BD human subjects characterized for suicidal behaviour and healthy controls were processed for proteomic analysis. In order to test the effect of lithium, each LCL was divided into two lines cultured for 7 days in media with and without LiCl (1.0 mmol/l). The proteome analysis highlighted 17 proteins differentially expressed in at least one of the comparisons or according to lithium treatment. These proteins are currently being identified by mass spectrometry. *Corresponding author Alessio Soggiu, PhD Department of Neurosciences B.B. Brodie, Section of Neurophysiology and Neurochemistry University of Cagliari, Monserrato (CA) [email protected] Phone: Fax: Herberth, M., et al., Peripheral profiling analysis for bipolar disorder reveals markers associated with reduced cell survival. PROTEOMICS, (1): p Pennington, K., et al., Prominent synaptic and metabolic abnormalities revealed by proteomic analysis of the dorsolateral prefrontal cortex in schizophrenia and bipolar disorder. Mol Psychiatry, (12): p Willour, V.L., et al., A genome-wide association study of attempted suicide. Mol Psychiatry, 2011.

46 2.34 PHYTOCHEMICALS RESVERATROL AND CURCUMIN AS POTENTIAL AGENTS IN THE PREVENTION AND THERAPY OF OVARIAN AND BREAST CANCERS Daniele Vergara a,d, Daniela Toraldo d, Piero del Boccio e, Damiana Pieragostino e, Pasquale Simeone a,e, Andrea Tinelli b, Stefania De Domenico c, Raffaele Acierno a,f, Giovanna Giovinazzo c, Andrea Urbani e, Angelo Santino c, Michele Maffia a,f a Laboratory of General Physiology, Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy b Department of Obstetrics and Gynaecology, Vito Fazzi Hospital, Lecce, Italy c Institute of Sciences of Food Production CNR-ISPA, Unit of Lecce, Italy d Laboratory of Clinical Proteomic, Giovanni Paolo II Hospital, ASL-Lecce, Italy e Research Centre on Aging (Ce.S.I), G. d Annunzio University Foundation,Chieti-Pescara Italy Considerable progress has been made in the development of novel therapeutic strategies in ovarian and breast cancer; however, conventional chemotherapies are often limited due to high toxicity and related side-effects, as well as the development of multidrug resistances. In this scenario, the multifactorial effects of dietary phytochemicals in blocking cancer initiation, promotion and progression have been known for several years. In particular, curcumin, a natural compound isolated from the plant Curcuma longa, and resveratrol, a polyphenol found in the skin of red grapes, show antiproliferative and proapoptotic effects against diverse tumors. Here, we investigated the antitumor activity of curcumin and resveratrol against a panel of breast and ovarian cancer cell lines and primary ovarian cells isolated from the ascitic fluid of ovarian cancer patients. Moreover, the potential use of these compounds as inhibitors of invasive processes was also investigated. By two-dimensional electrophoresis (2-DE) and MS/MS we found that curcumin and resveratrol inhibit cell proliferation through the regulation of the Akt / GSK / cyclin D1 pathway and induce apoptosis in a dose dependent manner. Additionally, our data provide new evidence of the inhibitory effect of resveratrol on growth factors-induced breast cancer cell transformation. The ability of these phenolic compounds to block cell proliferation could be a useful tool in the treatment of carcinoma tumors and beneficial for the prevention of cancer metastasis. Corresponding author prof. Michele Maffia. Laboratory of General Physiology, Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy. Fax: address: [email protected] 2.35 EFFECTS OF DEGENERATION-REGENERATION INDUCED BY NERVE CRUSH IN RAT GASTROCNEMIUS MUSCLE. Laura Barbalini 1, Daniele Capitanio 1, Chiara Fania 1, Michele Vasso 1,2, Roberta Leone 1, Isabelle Perroteau 3, Stefano Geuna 4, Cecilia Gelfi 1,2 1 Department of Sciences and Biomedical Technologies, University of Milan, Segrate (MI), Italy. 2 Institute of Bioimaging and Molecular Physiology, National Research Council, Segrate (MI), Italy. 3 Department of Animal and Human Biology, University of Turin, Turin, Italy. 4 Department of Clinical and Biological Sciences, University of Turin, Turin, Italy. Peripheral nerve lesions occur as a consequence of a variety of traumas and diseases inducing a dramatic muscle atrophy. Although transected peripheral nerves can spontaneously regenerate leading to functional recovery, axon regeneration requires a longer time during which the denervated muscles undergo atrophy. The required time hinder the functional muscle recovery after reinnervation. To monitor these events, 2-D DIGE and mass spectrometry were applied to profile the qualitative and quantitative differences in the proteome of rat gastrocnemius muscle during the process of denervation-reinnervation after sciatic nerve crush. The changes in protein expression levels were analysed at days: 1, 3, 7, 15, 30 after nerve crush, respectively, using non-crushed samples as controls. Results revealed 240 differentially expressed spots matched among the 6 groups. Statistical multivariate analysis highlighted 9 main spot clusters with a time-course expression pattern compatible with the degenerative-regenerative process. The major alterations were shown at time point 3 and 7, after which protein levels were progressively renormalized. After mass spectrometry identification, proteins were grouped in 11 functional classes, the majority of them were involved in energy transduction (8 spots), glycolysis (20 spots), transport (19 spots), stress response (13 spots). Furthermore, 19 spots were identified as structural components of the muscle cell. Next goal will be to define the contribution of these quantitative protein variations after merging data from mrna arrays on the same samples defining targets for the degeneration-reinnervation process. Corresponding author: Cecilia Gelfi Associated Professor Università degli Studi di Milano Via F.lli Cervi, Segrate (MI) [email protected] Tel Fax

47 3.2 PROTEOMICS TO EVALUATE THE EFFECT OF CRYOPRESERVATION ON POST-THAW STALLION SPERM Pamela Bonaccini1, Alessandro Gaviraghi2, Hani Husssein2, Luigi Bonizzi2, Stefano Romagnoli1, Maria Elena Falomo1, Paola Roncada3 1 Dip. Scienze Cliniche Veterinarie, Università degli Studi di Padova, Legnaro (PD); 2 DIPAV, Facoltà di Medicina Vetrinaria, Università degli Studi di Milano, Milano; 3 Istituto Sperimentale Italiano L. Spalllanzani, Milano The success of cryopreservation with stallion sperm is lower than that of bull (Watson, 2000). However, increased application of assisted reproductive technologies in the equine breeding industry has encouraged the use of frozen semen to preserve superior genetics. Stallion are commonly selected on the base of athletic performance record, pedigree and conformation. Freezability of semen is a term used to indicate survival rates of sperm populations by laboratory testing following a freeze-thaw cycle % of stallion have sperm with unacceptable freezability (Vidament et al., 1997). Over the years various freezing extenders were tested to improve the quality of frozen stallion semen, but there are lack of data and controversial theory. In addition, inter- and intra- stallion variations exist regarding freezability of sperm and in vivo fertility post-thaw (Salazar et al, 2011). In this study proteomic analysis was performed to compare sperm protein expression profile from seven stallion with different level of freezability. Comparative proteomic analysis showed expression of proteins related to high and low semen freezability. Some of that proteins are seminal plasma proteins, and we could hypothesize that they are involved in protection of sperm membranes during semen freezing procedures and against oxidative damage. Proteomics can provide a deeper understanding of protein functions in semen cryopreservation and can demonstrate the importance of post-translational modifications. Further large-scale proteomic studies will lead to the development of novel biomarkers that may allow for detection of semen abnormalities, essential for obtain higher reproductive efficiency and to ensure lower cost and time-loss by breeder. Bonaccini Pamela MVD Dip. Scienze Cliniche Veterinarie, Università degli Studi di Padova, Legnaro (PD) [email protected] Phone: References - Salazar JL, Teague SR, Love CC, Brinsko SP, Blanchard TL, Varner DD, Effect of cryopreservation protocol on postthaw characteristic of stallion sperm. Theriogenology, in press - Vidament M, Dupere AM, Julienne P, Evain A, Noue P, Palmer E, Equine frozen semen: freezability and fertility field results. Theriogenology 48: Watson PF The causes of reduced fertility with cryopreserved semen. Anim. Reprod Sci 6061:

48 3.3 OVOTRANSFERRIN AND GALLINACIN-9 FOUND IN SEMINAL PLASMA OF ROOSTER Annelisse Castillo1, Margherita Marzoni1, Simona Sagona1, Lorenzo Citti2, Silvia Rocchiccioli2, Isabella Romboli1, Antonio Felicioli1 1Dipartimento di Scienze Fisiologiche Sede Facoltà di Veterinaria, University of Pisa, Pisa, Italy; 2Istituto di Fisiologia clinica CNR of Pisa, Pisa, Italy Background: In birds, seminal plasma interacts with spermatozoa by stimulating sperm mobility (1,2). A deleterious effect on spermatozoa due to seminal plasma was evidenced in semen stored before use in artificial inseminations (3,4). This preliminary investigation intends identifying the rooster seminal plasma proteins using 2-DE and mass spectrometry. Methods: Semen were collected from four roosters, pooled and centrifuged in order to separate seminal plasma from spermatozoa and the supernatant frozen in liquid nitrogen. 50 µl of seminal plasma was suspended in 100 µl of PBS ph 7.4 with inhibitor of protease and centrifuged. The supernatant was mixed with 10 drops of TCA 10% and placed in ice for 45 min before new centrifugation. The pellet was resuspended in 250 µl of rehydration buffer. 100 microgram of seminal plasma proteins were loaded on each 11cm Immobiline DryStrip, ph SDS-PAGE was performed using self-cast 12% polyacrylamide gels. The detected spots were analysed by MALDI TOF TOF mass spectrometry. Results: The resulting gels were analysed by PDQuest software. 83 spots were detected. Ten proteins were successfully identified by mass spectrometry and the others are under analysis. Among the identified proteins, ovotransferrin was strongly expressed while gallinacin-9 was moderately expressed. In Gallus gallus, ovotransferrin was previously reported to be expressed in oviduct and egg and Gallinacin-9 was expressed in both female and male reproductive organs of (5,6,7,8,9,10). The presence of ovotransferrin and gallinacin-9 in seminal plasma of roosters is here reported for the first time. Corresponding Author Antonio Felicioli Dipartimento di Scienze Fisiologiche, sede di Medicina Veterinaria, viale delle Piagge 2, 56100, Pisa [email protected] Phone Fax References 1) Cornwallis CK, O'Connor EA. Sperm: seminal fluid interactions and the adjustment of sperm quality in relation to female attractiveness. Proc. R. Soc. B. 2009;276: ) Froman DP, Wardell JC, Feltmann AJ. Sperm mobility: Deduction of a model explaining phenotypic variation in roosters (Gallus domesticus). Biol Reprod. 2006;74: ) Blesbois E, De Reviers M. Effect of different fractions of seminal plasma on the fertilizing ability of fowl spermatozoa stored in vitro. J Reprod Fertil. 1992;95: ) Douard V, Hermier D, Magistrini M, Blesbois E. Reproductive period affects lipid composition and quality of fresh and stored spermatozoa in Turkeys. Theriogenology 2003;59: ) Das SC, Isobe N, Yoshimura Y. Expression of Toll-like receptors and avian β-defensins and their changes in response to bacterial components in chicken sperm Poultry Sci. 2011;90: ) Jonchère V, Réhault-Godbert S, Hennequet-Antier C, Cabau C, Sibut V, Cogburn LA, Nys Y, Gautron J. Gene expression profiling to identify eggshell proteins involved in physical defense of the chicken egg. BMC Genomics. 2010;11: 57. 7) Michailidis G, Avdi M. Transcriptional profiling of gallinacins antimicrobial peptides in the chicken reproductive tract and embryos. J Biol Res Thessalon. 2010;14: ) Milona P, Townes CL, Bevan RM, Hall J. The chicken host peptides, gallinacins 4, 7, and 9 have antimicrobial activity against Salmonella serovars. Biochem Bioph Res Co. 2007;356: ) Xiao Y, Hughes A.L, Ando J, Matsuda Y, Cheng J.-F, Skinner-Noble D, Zhang G. A genome-wide screen identifies a single beta-defensin gene cluster in the chicken: implications for the origin and evolution of mammalian defensins. BMC Genomics. 2004;5:56. 10) Gautron J, Hincke MT, Panheleux M, Garcìa-Ruìz JM, Boldicke T, Nys Y. Ovotransferrin is a matrix of the hen eggshell membranes and basal calcified layer. Connect Tissue Res. 2001(42);4: IL-8 TRANSCRIPTIONAL MACHINERY IN RESPIRATORY EPITHELIAL CELLS EXPOSED TO PRO-INFLAMMATORY STIMULI SPECIFIC OF CF LUNG DISEASE Carla Iannone*, Maria Monti*, Valentino Bezzeri, Monica Borgatti#, Roberto Gambari#, Giulio Cabrini and Piero Pucci* *Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II; Laboratorio Patologia Molecolare, Azienda Ospedaliera Universitaria di Verona; #Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Ferrara CEINGE Biotecnologie Avanzate, Napoli. The hallmark in CF airway pathology is a characteristic neutrophil (PMN)- dominated inflammation. PMNs, continuously activated by bacterial products, release Reactive Oxygen Species and proteases, that are the major contributors of CF lung tissue injury Reduction of PMN infiltrates is now considered a CF-specific anti-inflammatory target. However, the precise mechanisms of transcription of Interleukin 8 (Il-8), the main chemokine released by epithelial respiratory cells to recruit PMNs in CF lungs, are not fully understood, thus limiting the possibility of tailoring novel well-targeted anti-inflammatory molecules. The general aim of this research project is to fully explore the transcriptional machinery of IL-8 in respiratory epithelial cells exposed to pro-inflammatory stimuli specific of CF lung disease. The region -180/-21 of IL-8 promoter was investigated by functional proteomics approach in order to identify proteins and/or protein complexes able to bind this DNA sequence. The -180/-21 oligonucleotide fragment was synthesised with a biotin tag and then immobilised on streptavidin derivatised magnetic beads. A random biotinilated oligonucleotide with similar length was used as control. The protein nuclear extract from CF bronchial epithelial IB3-1 pre-treated with P. aeruginosa for activation of pro-inflammatory stimuli was incubated with the specific oligonucleotide bait. The protein ligands specifically retained on the beads after extensive washing cycles were eluted by Laemli buffer, boiled and fractionated by SDS PAGE. Protein bands, stained by Colloidal Coomassie, were excised from the control and the sample lanes and digested by trypsin. The resulting peptide mixtures were analysed by nanolcmsms and the corresponding proteins identified by MASCOT database search software. Several proteins were identified. Corresponding Author Carla Iannone, PhD student, Department of Organic Chemistry and Biochemistry and CEINGE Biotecnologie Avanzate s.c.a r.l., University of Naples, Federico II [email protected] phone: fax: We gratefully acknowledge contributions from Rete Nazionale di Proteomica, Progetto FIRB RBRN07BMCT, Italian Human Proteomenet; Fondazione Italiana per la Ricerca sulla Fibrosi Cistica.

49 3.7 DESIGN OF A BIOINFORMATICS ALGORITHM FOR THE ASSESSMENT OF MOUSE GUT PHYLOTYPES IDENTIFIED BY METAPROTEOMIC APPROACH Lorenza Putignani a, Stefano Levi Mortera b, Federica Del Chierico a, Pamela Vernocchi a,c, M. Manuela Rosado d, Rita Carsetti d, and Andrea Urbani b,e a Microbiology Unit, Immunology Research Area, Rome, Italy; b Department of Internal Medicine, University of Rome Tor Vergata, Rome, Italy; c Department of Food Science, Alma Mater Studiorum- University of Bologna, Italy; d Immunology Research Area, B-cell development Unit, Immunological Diagnosis Unit; e European Center Brain research, IRCCS-Fondazione S. Lucia, Roma, Italy Onset and shaping of gut microbiota rapidly evolve after birth and diverge, in an individual-dependent way, under the effect of spatial and temporal variability determinants. A full comprehensive description of human gut phylotypes is essential to highlight gut homeostasis and perturbation. However, classical microbiology is underpowered by its inability to provide unbiased representation of gut microbiota. In order to design a bioinformatics protocol able to interpret the massive data arisen from a metaproteomics approach, a mouse gut model was exploited. Mucosal biopsies from 16 Balb/c and Rag2KO mouse babies were collected and processed as follows: after homogenization, biopsies were inoculated in Brain Heart Infusion (BHI) medium and ON incubated under gentle agitation. Culture was centrifuged and the pellet stored at -80 C until peptide triptic digestion. LC-MS was performed with a Proxeon EASY-nLCTM and a Bruker amazone Ion Trap mass spectrometer equipped with a nanoflow ESI Sprayer. Mascot Distiller software was used to process data, under AutoMSn mode and for database searching (Swiss-Prot, bacteria taxonomy). Data interpretation algorithm consisted of the following steps: i) enumeration of single peptide associated to each taxon unit (TU, strain phylotype); ii) grouping of peptides linked to each TU; iii) enumeration of protein IDs for each TU; iv) grouping of protein IDs linked to each TU, filtered by nomenclature; v) screening of equiprobable TU by microbiological significance (e.g., host specificity) and selection of superior TU (species phylotype). Fast scan rate and high sensitivity of ion trap are particularly suitable for this wide range identification approach. 4.1 COMBINING TOP DOWN AND BOTTOM UP ANALYSES IN A SINGLE LCMS EXPERIMENT Reinaldo Almeida Zum Kapellenwald Arnsberg Germany In this work we use bottom up and top down analyses by combining online LCMS with nanoesi infusion for the identification of proteins and characterizing their post-translational modifications during the brewing process. The intact mass measurement and primary sequence determination was performed by online UPLC-MS(MS) with simultaneous fraction collection. The top down affords the post-translational modification observation after the UPLC-MS run by automated nanoesi infusion of the previously collected fractions. Since nanoesi consumes just a small amount of the analyte, proteolytic digestion was performed on the remaining sample for bottom up analyses. This strategy allows us to full characterise proteins involved during brewing, by intact mass determination, PTM characterisation and the corresponding peptide sequence coverage in a single LCMS experiment. The combination of intact protein chromatography by UPLC with a post column splitting system, showed equal separation power and signal to noise ratio, in comparison with the standard non splitting set up. During the online top down analyses the intact mass could be determined by deconvoluting the charge state envelop. However many proteins have been detected but not identified or with low confidence due to the limited time during the elution. These proteins where then targeted during the offline analyses of the previous collected fractions for fop down MS/MS, but also for intact mass determination of very low abundant species. The offline analyses allowed an unlimited averaging capability and optimisation due to the low sample consumption of nanoesi as well as the specific targeting of post translational modifications. Each protein fraction was then digested and reanalysed by offline bottom up, giving again unlimited time for optimising the condition and averaging during the analyses of the corresponding peptides. The good UPLC separation of the intact proteins lead that the peptides identified in each fraction can just correspond to the proteins found by the top down approach previously, adding another confidence level to the analyses. Furthermore the complexity in each fraction is reduced in a way that no 2 dimension chromatography run is needed. The combined top down and bottom up results facilitated the interpretation of unknown proteins as well as post translational modifications. [email protected] Pamela Vernocchi, Microbiology Unit, Immunology Research Area, Children Hospital and Research Institute Piazza Sant Onofrio, 4, Rome, Italy Phone: Fax: s: [email protected] [email protected]

50 4.2 TOP-DOWN RP-HPLC-ESI-MS PLATFORMS FOR THE DETECTION OF THE INTACT NATURALLY OCCURRING HUMAN SALIVARY PROTEOME. Massimo Castagnola*1,, Federica Iavarone1, Chiara Fanali1, Rosanna Inzitari1, Federica Vincenzoni1,, Tiziana Cabras2Barbara Manconi2, Maria T. Sanna2, Alessandra Olianas2, Elisabetta Pisano2, Roberto Boi2, Sonia Nemolato3, Alberto Vitali4, Emanuele Scarano5, Antonella Fiorita5, Giulio C. Passali5, Armando Manni6, Massimo Cordaro6,Gaetano Paludetti5, Gavino Faa3, Irene Messana2. 1Istituto di Biochimica e Biochimica Clinica, 5Istituto di Otorinolaringoiatria, 6Ist. di Clinica Odontoiatrica, Facoltà di Medicina, Univ. Cattolica, Roma, Italy. 2Dip. di Scienze della Vita e dell Ambiente e di 3Citomorfologia, Univ. di Cagliari, 4Ist. per la Chimica del Riconoscimento Molecolare, CNR Roma. Proteomic platforms can be classified in bottom-up (such as shot-gun proteomics) which analyses the sample following its proteolytic (usually trypsin) digestion, and top-down strategies, which analyse the intact, naturally occurring proteome. Bottom-up platforms are characterised by a high-throughput because they can identify variations in the amount of any protein, regardless its dimension. Nonetheless, information on PTMs can be lost, especially those regarding naturally occurring cleavages. Topdown platforms usually cannot cover vast proteomes (i.e. excluding proteins insoluble in acidic milieu). However, they can disclose subtle structural variations occurring during protein maturation undetectable in shot-gun strategies. Moreover, they allow label-free relative quantifications in limitless number of samples by the application of XIC (exctracted Ion Current) searches or SRM and MRM measurements. A repertoire of 236 naturally occurring protein and peptide masses detectable by RP-HPLC-ESI-MS analysis on the acidic soluble solution of human whole saliva will be presented. The top-down platform, reported in a devoted flow chart, allowed the identification of 213 components, 23 of them still pending for a structural identification. The table shows the peptides and proteins identified and it is organized in different columns reporting average and mono-isotopic masses, m/z values useful for XIC searches, elution time in conventional (C8 column using 0.5% TFA as ionpairing and ionization agent) RP-HPLC separations, PTMs, origin, specific variations observed in physiological and pathological conditions as well as the m/z values used for XIC searches. The table can be used as a reference for anyone would experience top-down RP-HPLC-ESI-MS proteomic platforms of the human salivary proteome and it is obtainable in Excel format at the address of the corresponding author. 4.3 THE ORBITRAP TECHNOLOGY: INCREASED ANALYTICAL PERFORMANCES FOR THE ANALYSIS OF PEPTIDES AND PROTEINS. Martin Zeller; Catharina Crone; Carmen Paschke; Mathias Mueller; Eugen Damoc; Eduard Denisov; Alexander Makarov; Dirk Nolting; Bernard Dealnghe; Thomas Moehring Thermo Fisher Scientific, Bremen, GERMANY Getting a comprehensive view of the protein content of a complex sample is still a challenge in proteomics, getting that done in one analytical run even more. This work will describe new analytical platforms and software with increased performances in terms of scan speed, resolution and sensitivity. A tryptic digest of a whole SILAC labeled HeLa cell lysate was measured on a new Thermo Fisher Scientific benchtop quadrupole Orbitrap instrument coupled to a Thermo Fisher Scientific Easy nano-lc. More than HCD spectra were acquired. The raw files were analyzed using Mascot, SEQUEST and X!Tandem. The statistical significance of the identifications was done by applying Percolator to the primary target decoy results of the individual search engines. This resulted in more than peptides and more than 2000 proteins identified with less than 1% FDR. Enzymatically degraded E.coli total protein extract was analyzed on a modified Thermo Fisher Scientific LTQ Orbitrap Velos instrument including a compact Orbitrap analyzer and improved pre-amplifier which results in a higher scan speed and improved dynamic range and sensitivity. Proteome Discoverer 1.2 was used for database searching and result validation. The higher scan speed in combination with the better sensitivity results in a higher number of protein and peptide identifications compared to the standard LTQ Orbitrap Velos instrument using the same sample and LC conditions. More precursor ions of low abundant compounds get over the limit of detection and the faster scan rate allows also triggering those precursor ions for MS/MS. Claire Dauly LSMS Proteomics Marketing Specialist Scientific Instrument - Thermo Fisher Scientific 16 Avenue du Quebec - SILIC 765, COURTABOEUF CEDEX, France [email protected] Corresponding author: Massimo Castagnola (Full Prof.) Ist. di Biochimica e di Biochimica Clinica, Facoltà di Medicina, Università Cattolica Largo Francesco Vito Roma, Italy [email protected] or [email protected] Tel. and Fax: or This work was supported by Università Cattolica, Università di Cagliari, Istituto Scientifico Internazionale Paolo VI, MIUR, Consiglio Nazionale delle Ricerche, Regione Autonoma Sardegna e Fondazione Banco di Sardegna.

51 4.7 TARGETED TOP-DOWN PROTEOMIC ANALYSIS OF CEREBROSPINAL FLUID FROM AMYOTROPHIC LATERAL SCLEROSIS PATIENTS Marta Vilaseca1, Claudio Diema1, Núria Omeñaca1, Alex Campos3, Eliandre de Oliveira3, Mark Baumert5, Kees Vlak5, Joan Guinovart2, Jacques Borg4 1 Mass Spectrometry Core Facility, Institute for Research in Biomedicine, Barcelona, Spain 2 Institute for Research in Biomedicine, Barcelona, Spain 3 Proteomic Platform, Scientific Technical Services, Barcelona Science Park, Spain 4 Faculty of Medicine Jacques Lisfranc, Jean Monnet University, Saint-Étienne, France 5 Advion BioSciences, Harlow, Essex, UK Introduction Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects motoneurons in the spinal cord and brain. Diagnosis is achieved with clinical evaluation, as well as electromyography and neuroimaging. However, a significant number of patients are misdiagnosed or diagnosed after a long delay. Therefore, it is crucial to find potential biomarkers to improve early and reliable diagnosis. Previous studies have reported differentially expressed proteins in various neurodegenerative diseases, as well as posttranslational modifications (PTM). Using a Bottom-Up analysis and isobaric quantification of cerebrospinal fluid (CSF) from ALS patients, we recently identified a panel of differentially expressed proteins. In the present study we applied Top-Down proteomics to examine modifications of these proteins in CSF samples. Methods Top-Down analysis was performed by online LC-nanoESI-MS coupling on a LTQ-FT Ultra (ThermoScientific) with simultaneous fraction collection using the Triversa Nanomate (Advion BioSciences). A BioSuite pphenyl 1000 (Waters) 10 m RPC 2.0 x75 mm column with a 5-80% ACN gradient over 60 min (100 l/min) was used for intact protein separations. Protein MS/MS analyses were performed off-line, using the Nanomate, by applying CID or ECD and/or IRMPD fragmentation in order to determine the molecular weight of a high number of protein fragments. Complementary analysis of tryptic digested proteins of some of the fractions collected was performed by a Bottom-Up approach. Top-Down and Bottom-Up data were processed with ProsightPC 2 and Sequest (Bioworks 3.2; Thermo Scientific), respectively. 4.8 ULTRAHIGH-PERFORMANCE NANO LC-MS/MS ANALYSIS OF COMPLEX PROTEOMIC SAMPLES Steffen Liedtke,1 Evert-Jan Sneekes,2 Bjorn de Haan, 2 and Remco Swart2 1Dionex Corporation, Germering, Germany 2Dionex Corporation, Amsterdam, The Netherlands Determination of the proteome and identification of biomarkers are required to monitor dynamic changes in living organisms and predict the onset of an illness. One popular method to tackle contemporary proteomic samples is called shotgun proteomics, in which proteins are digested, the resulting peptides are separated by high-performance liquid chromatography (HPLC), and identification is performed with tandem mass spectrometry. Digestion of proteins typically leads to a very large number of peptides. For example, digestion of a cell lysate easily generates 500,000 peptides. The separation of these highly complex peptide samples is one of the major challenges in analytical chemistry. The main strategy to improve the efficiency of packed columns is either to increase column length or to decrease the size of the stationary phase particles. However, to operate these columns effectively, the LC conditions need to be adjusted accordingly. Naturally, the on-line coupling to MS systems has to be taken into account in the optimization process. Here, the authors report on the performance of nano LC columns operating at ultrahigh pressure. The effects of column parameters (particle size and column length) and LC conditions (gradient time, flow rate, column temperature) were investigated with reversed-phase (RP) gradient nano LC. High-resolution LC-MS separations of complex proteomic peptide samples are demonstrated by combining long columns with 2 µm particles and long gradients. The effects of LC parameters on performance and the influence on peptide identification are discussed. Franco Abballe Support Manager Dionex S.p.A. a part of Thermo Fisher Scientific Via XXV Aprile San Donato Milanese (Mi) Fax [email protected] Preliminary data We focused our Top-Down studies on a few proteins that were found to be differentially expressed, like microglobulin, which was down-regulated, or apoliprotein, which was up-regulated in ALS samples compared to controls. Fractions of interest were analyzed at high resolution in full MS (R= ). CID was performed in the ion trap with fragment detection in the LTQ-FT. CID, ECD and/or IRMPD data were acquired with scans averaging in order to increase signal to noise ratio and to obtain more informative spectra. PTM isomers were also quantified by comparing the ratios of fragment ion abundance produced by MS/MS. Our data show several phosphorylation and methylation patterns for these potential biomarkers for ALS. More specifically, modified proteins were studied with the aim of using phosphorylation and/or other PTM as a diagnosis tool. Novel aspect Implementation of Top-Down methodology for PTM pattern analysis of potential ALS biomarkers in CSF. Kees Vlak, [email protected]

52 4.9 TWO DIMENSIONAL SEC // RP CAPILLARY LC FOR TOP-DOWN PROTEOMICS ANALYSIS Robert van Ling1, Franco Abballe 2, Evert-Jan Sneekes3, Perry van Paassen3, Macro Karsten3, Wim Decrop3, Remco Swart3 1Thermo Fisher Scientific, Olten, Switzerland 2Thermo Fisher Scientific, Milan, Italy 3Thermo Fisher Scientific, Amsterdam, The Netherlands Top-down proteomic LC-MS aims at investigating protein structure and post-translational modifications (PTM) through liquid phase separation and MS analysis of intact proteins. The advantage of top-down approaches over bottom-up approaches is the absence of a proteolytic cleavage step. This keeps all protein information within one molecule and does not multiply sample complexity. However, working with intact proteins introduces challenges for the analytical technology used. Proteins are more difficult to separate than peptides using standard LC methods and more difficult to study by MS. Recently, different electron based dissociation techniques, such as ECD and ETD have been successfully applied to intact proteins to obtain sequence and PTM information. The successful application of these techniques in tandem mass spectrometry for protein mixtures requires a separation step but powerful liquid chromatographic methods for proteins are scarcely published and need to be developed and optimized for direct interfacing with mass spectrometry. In this study we have developed a two dimensional capillary scale LC method for the separation of intact proteins. Proteins are separated and fractionated on a capillary size exclusion column. Next, proteins contained in the size fractions are separated on reversed phase capillary monolithic columns prior to UV and MS detection. The method has been optimized with respect to SEC fractionation and RP gradient conditions for standard proteins and complex mixtures. Franco Abballe Support Manager Dionex S.p.A. a part of Thermo Fisher Scientific Via XXV Aprile San Donato Milanese (Mi) Fax [email protected] 4.10 OPTIMIZING PARTICLE SIZE AND COLUMN LENGTH, WHAT IS THE BEST WAY TO UTILIZE NANO UHPLC IN PROTEOMICS? Robert van Ling1, Franco Abballe 2, Evert-Jan Sneekes3, Macro Karsten3, Wim Decrop3, Remco Swart3 1Thermo Fisher Scientific, Olten, Switzerland 2Thermo Fisher Scientific, Milan, Italy 3Thermo Fisher Scientific, Amsterdam, The Netherlands The development of UHPLC capabilities on nano LC scale has opened up new ways to provide proteomics researchers with the peak capacity they need for their work. UHPLC principles involve the use of smaller particles allowing faster analysis and/or better resolution; it is usually focused on throughput in small molecule analysis. However in proteomics analysis speed is of lesser concern compared to peak capacity. The increased pressures in UHPLC can be used to support increased column lengths as well. With 25 and even 50 cm columns being implemented in routine applications, the 1 meter barrier has become visible. Here we utilize the full potential of nano UHPLC to determine the effects of smaller particles and column length on protein identification. In this study we used different nano columns at the highest possible pressure to separate a complex tryptic digest. The particle size and the length of the column were varied to create columns that operate at upper end of the system pressure limits. All columns were operated at typical nano flowrates of nl/min and directly interfaced (ESI) with a mass spectrometer. The gradient length was varied to determine the optimal gradient for each column length. Peak capacity, sequence coverage and protein identifications were used to determine the efficiency of the separation. A smaller particle, often associated with UHPLC, will generate more backpressure. Therefore a 50 cm long column packed with 2 µm particles already operates at bars, which is the upper pressure range of the used nano LC instrument. Increasing the particle size to 3 µm allowed column lengths of 1 meter and operation in the same pressure range. Higher temperatures and smaller particles allow even longer columns. When using these extremely long columns, gradients have to be optimized. Running a too steep gradient will not use the full potential of the column. Only when the gradient length is shallow enough the full length of the column could be utilized and peak capacities approaching 1000 were reached. Franco Abballe Support Manager Dionex S.p.A. a part of Thermo Fisher Scientific Via XXV Aprile San Donato Milanese (Mi) Fax [email protected]

53 4.11 EXPLORATION OF PH-GRADIENT, ION- EXCHANGE CHROMATOGRAPHY FOR HIGH- RESOLUTION PROTEIN SEPARATIONS IN BIOTECHNOLOGY AND PROTEOMICS Wim Decrop, Marie-Jeanne Olivo, Evert-Jan Sneekes, and Remco Swart Dionex Corporation, Amsterdam, The Netherlands Ion-exchange chromatography (IEC) is a versatile separation technique for profiling the charge heterogeneity of biotherapeutic proteins, including monoclonal antibodies. Despite good resolving power and robustness, ionic-strength-based ion-exchange separations are product specific and time consuming to develop. Although salt gradients are more commonly applied, the utilization of ph gradients can provide significant advantages such as: 1) improved separation resolution; 2) lower salt concentration in collected fractions; and 3) the possibility to correlate the protein isoelectric point (pi) data with elution profiles. Recently, the application of ph-gradient IEC has been described for the separation of standard proteins1 and monoclonal antibodies.2,3. The work shown here describes the application of ph-gradient IEC as compared to salt-gradient IEC for the separation of proteins from various sources. High-resolution separations of a monoclonal antibody and its isoforms were achieved using a new, nonporous, strong cation-exchange resin. Results were compared to those obtained with salt-gradient IEC. Complex protein mixtures typically found in proteomics were separated with ph-gradient IEC. Developed methodology was validated for ph profile shape and precision, retention-time precision, peak capacity, and robustness towards sample solvent composition. Franco Abballe Support Manager Dionex S.p.A. a part of Thermo Fisher Scientific Via XXV Aprile San Donato Milanese (Mi) Fax [email protected] References 1. Ahamed, T. et al., Selection of ph-related Parameters in Ion- Exchange Chromatography Using ph-gradient Operations. J.Chromatogr., A 2008, 1194 (1), SET-UP FOR HUMAN SERA MALDI PROFILING: THE CASE OF RHEPO TREATMENT Enrica Torretta 1, Chiara Fania 1, Michele Vasso 1,2, Carsten Lundby 3, Cecilia Gelfi 1,2 1 Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi di Milano, Milan, Italy 2 Istituto di Bioimmagini e Fisiologia Molecolare, C.N.R., Segrate, Milan, Italy 3 Copenhagen Muscle Research Centre, University of Copenhagen, Copenhagen, Denmark The implementation of high-throughput technologies based on qualitative and quantitative methodologies for the characterization of complex protein mixtures is increasingly required in clinical laboratories. MALDI profiling is a robust and sensitive technology even though the serum high dynamic range imposes some limitations, preventing detection and identification of less abundant species. Efforts to increase the MALDI profiling detection ability are needed. A set-up has been performed for recombinant human erythropoietin (rhepo) monitoring in serum analyzing the effects of two commercially available columns (MARS Hu7 and Hu14) for immunodepletion, and two matrices (α-cyano-4-hydroxycinnamic acid and 2,4 -dihydroxyacetophenone) for spectra quality improvement. Immunodepletion skills of both columns were determined by 2D- DIGE, which precisely revealed the efficacy of Hu14 in protein removal and in serum dynamic range decrement. After optimization of the type of matrix and sample dilution, these new efficient conditions were used for serum profiling of ten healthy subjects before and after rhepo treatment. The principal component analysis indicates that combination of Hu14 column and 2,4 -dihydroxyacetophenone matrix increases data quality allowing to discriminate between treated and untreated samples, making serum MALDI profiling suitable for clinical monitoring of rhepo. Cecilia Gelfi, Professor, Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi di Milano, Milan, Italy; via Fratelli Cervi 93, Segrate, Milan, Italy [email protected] Fax: Reference: Fania C., Vasso M., Torretta E., Robach P., Cairo G., Lundby C., Gelfi C., Setup for human sera MALDI profiling: The case of rhepo treatment, Electrophoresis, in press. 2. Farnan, D.; Moreno, G. T. Multi-Product High-Resolution Monoclonal Antibody Charge Variant Separations by ph Gradient Ion-Exchange Chromatography. Anal. Chem. 2009, 81 (21), Rea, J. C.; Moreno, G. T.; Lou, Y.; Farnan, D. Validation of a ph Gradient-Based Ion-Exchange Chromatography Method for High- Resolution Monoclonal Antibody Charge Variant Separations, J. Pharm. Biomed. Anal. 2011, 54 (2), Kaliszan, R.; Wiczling, P.; Markuszewski, M. J. ph Gradient High- Performance Liquid Chromatography: Theory and Applications. J.Chromatogr., A 2004,1060, Ahamed, T. et al., ph-gradient Ion-Exchange Chromatography: An Analytical Tool for Design and Optimization of Protein Separations. J.Chromatogr., A 2007, 1164, Tsonev, L. I.; Hirsh, A. G. Theory and Applications of a Novel Ion Exchange Chromatographic Technology Using Controlled ph Gradients for Separating Proteins on Anionic and Cationic Stationary Phases, J.Chromatogr., A 2008, 1200,

54 5.2 CHARACTERIZATION OF ANTENNA ISOFORMS FROM SPINACH LEAVES GROWN UNDER DIFFERENT LIGHT CONDITIONS Federica Gevi, Anna Maria Timperio, Sara Rinalducci, Maria Giulia Egidi, Lello Zolla Department of Environmental Sciences, University of Tuscia, Viterbo, Italy The major light-harvesting complex (LHC-II) of higher plants plays a crucial role in capturing light energy for photosynthesis and in regulating the flow of energy within the photosynthetic apparatus. Native LHC-II isolated from plant tissue consists of three isoforms, Lhcb1, Lhcb2, and Lhcb3, which form homo- and heterotrimers. Usually plants are exposed to environment where light and temperature conditions are largely variable. They have evolved several mechanism of acclimation. Among all the environmental parameters light intensity has a major influence on plants life. During light stress at 100 µe m-2 s-1 plants must balance the excitation of the two photosystems (Allen et al. 2001, Timperio et al. 2005) and so there is a migration of light-harvesting complex type 2 (Lhcb2) from grana to PSI containing stroma exposed membranes. Changes in the association-dissociation state of LHCII are the regulatory mechanism that optimize light-harvesting function under different stress condition. In this communication we want to investigate the behaviour of antenna proteins (Lhcb) during different acclimatation. For this purpose we used a microrotofor (Biorad) that avoids detergent and we obtained the antenna proteins in their native form. Separation of complexes is based on the protein s real physicochemical properties which inevitably change when dye is added. The ph of each native complex was also determined: this is the case of various complexes of LHCII trimers, which have different ph but similar molecular masses. A reverse-phase HPLC method on line with mass spectrometry allowed us to have a qualitative and quantitative analysis of Lhcb proteins. Correspondig Author: Timperio Anna Maria Assistant Professor Department of Environmental Sciences University of Tuscia Proteomics and Genomics Lab Largo dell Università snc Viterbo, Italy [email protected] tel fax References: Timperio AM, Zolla L. J Biol Chem. 2005;280(32): Allen JF, Forsberg L. Trends Plant Sci. 2001;6:

55 5.5 SALT STRESS RESPONSES IN PINUS HALEPENSIS Lomaglio Tonia1, Satriani, A.2, Verrillo, F.1, Rocco, M.1 1Department of Science for Biology, Geology and Environment, University of Sannio, Benevento, Italy 2CNR-Institute of Methodologies for Environmental Analysis, Potenza, Italy Metapontum Forest Reserve is an artificial formation located between mouths of Bradano and Basento river, it is composed prevalently of Aleppo pine (Pinus halepensis). In recent years, the Metapontum coast is characterized by a strong erosive process which has really removed the dune behind the beach moving in the inland and causing the decline of the historical pinewood. This negative effect on plant was induced by an increase in soil salinity, which is one of the major abiotic stresses. A clear understanding of the molecular mechanisms involved in plants response to salt stress is fundamentally important for plant biology. The salinity soil causes broad variety of physiological and biochemical processes, as oxidative damage, also has a negative effect on energy metabolism, which unavoidably resulted in a decreased ATP production through photophosphorylation and, thus, affected the Calvin cycle in photosynthesis. A proteomic approach was utilized to identify key protein which result to be directly responseve to salt stress. Total proteins were extracted from the leaves by a combination of TCA acetone and phenol, and separated by two-dimensional gel electrophoresis at ph 5-8. Spots were stained with Comassie Brilliant Blue and analyzed with the software PDQuest 8.0 (Bio-Rad) to identify differentially expressed polypeptides. Preliminary analysis revealed around 29 differentially expressed proteins, and they were sequenced by MALDI TOF and LC-MS/MS. Sequenced spots were classified in different functional classes. Rocco Mariapina Dr. Mariapina Rocco Department of Science for Biology, Geology and Environment, University of Sannio Via Port Arsa, 11, Benevento, Italy [email protected] phone:0824/ fax:0824/ PROTEOMICS OF THE RIPENING FRUIT IN OLEA EUROPAEA Linda Bianco1, Luciana Baldoni2, Fiammetta Alagna2, Christine Finnie3, Birte Svensson3, Silvia Mazzuca4 and Gaetano Perrotta1 1 - ENEA CR Trisaia, Rotondella (MT) Italy 2 - CNR-IGV Perugia - Italy 3 - EPC System biology DTU, Lyngby Danmark 4 - Dip. Ecologia Università della Calabria Rende (CS) Italy Maturation is a physiological process, which has a remarkable impact on the modulation of the biosynthesis of compounds affecting the quality traits of olive fruits, influencing as well the final composition of olive oil. Protein extracts from fruits of cv. Coratina harvested at 45, 110 and 150 days after flowering were compared by 2DE. Statistical analysis elaborated with ProGenesis SameSpots software revealed 247 protein spots differentially accumulated during ripening. PCA showed complete separation among the ripening stages. The first PCA component explains, in fact, 72% of the variance, indicating that the stage of ripening is the largest source of variation. Most of differentially accumulated protein spots appear as train of spots, likely representing proteins with different degrees of post-translational modifications. These protein modifications would not ordinarily have been found using the sole genomic/ transcriptomic approaches, thus reinforcing the utility of proteomics. Among the differentially accumulated protein spots, about 220 were manually excised from the gel, subjected to trypsin digestion and analyzed by MALDI TOF-TOF mass spectrometry. Database searching of collected spectra and data analysis are currently under investigation. The protein spots characterized to date revealed some proteins supposed to play a pivotal role during fruit ripening, providing a widespread picture of metabolic and physiological bases of ripening process and oil accumulation in olive. This work is being carried out in the frame of a larger genomics survey of Olea europaea; we expect that progress in genomic characterization will in parallel reflect improvements in protein identifications and related gene functions. This work is supported by the Italian Ministry of Agriculture (MIPAF) Project OLEA Corresponding Author Gaetano Perrotta PhD ENEA (Agenzia nazionale per le nuove tecnologie, l energia e lo sviluppo economico sostenibile) S.S. 106 Jonica km 419, Rotondella (MT) [email protected]

56 Late Abstract PROTEOMIC CHARACTERIZATION OF MICRODIALYSIS HUMAN ADIPOSE TISSUE Nunno M.G. 1, Rocchetti M.T. 1, Murdolo G. 2, Grandaliano G. 3, Ranieri E. 4, Gesualdo L. 5, Cincione I. 4 1 Interdepartmental Research Center BIOAGROMED - University of Foggia, Foggia, Italy; 2 Dipartment of Internal Medicine, Endocrine and Metabolic Unit University, Perugia, Italy; 3 University of Foggia Nephrology - Department of Biomedical Sciences, Foggia, Italy; 4 University of Foggia Clinical Pathology Department of Biomedical Sciences, Foggia, Italy; 5 University of Bari, Renal, Dialysis and Transplant Unit, Department of Emergency and Organ Transplant, Bari, Italy Background: Microdialysis is an in vivo technique to study metabolism in different tissues unusually not easily accessible. The main advantage is that it can be used safely with low-grade invasiveness, allowing continuos sampling over prolonged periods of time from specific tissue without performing any biopsy. A microdialysis-based sensor can be placed in the subcutaneous adipose tissue, in order to not interfere with everyday activities. The aim of the present study was to identify early signs of damage at the onset of diabetes mellitus by analysing differential protein expression in adipose subcutaneous microdialysate (microfiltrate) in obese patients with or without diabetes mellitus (DM). As far as we know, this is the first investigation of human adipose tissue microdialysis by proteomic approach. First, we standardized a protocol for the collection, pre-analytical treatment and analysis of microfiltrate by two dimensional gel electrophoresis (2DE) combined with MALDI-TOF/MSMS. Methods: About 1 ml of microfiltrate was obtained from obese patients by using microdialysis catheter which consist of an inner tube and an outer tube with a semipermeable membrane. The catheter is placed in the interstizial space of the tissue of interest and the inner tube is continuously perfused, for three hours, with a perfusion fluid containing 0,9% saline without albumin. During the return through the outer tube an exchange of compounds between the tube and the interstitial space takes place over the semi permeable membrane. The obtained microfiltrate was concentrated by Amicon filter devices (3 kda cut off) and resuspended in a minimal amount of solubilization buffer appropriate for 2DE analysis. Proteins isoelectrofocusing was carried out using 7 cm IPG strip at ph 3-10 linear range at 30 kvolt hour total produced by overnight run. The second dimension SDS-PAGE was carried out on home-made polyacrylamide (12%) slab gels in SDS-PAGE running buffer at a constant voltage of 200 V. Analytical 2-DE gels were commasie blue colloidal stained. Samples were run in triplicate. Results: We obtained high-resolution 2-DE map of obese patient microfiltrate with BMI of 47.8, (figure 1). Image analysis (ImageMaster 2D Platinum) of 3 gels replicates detected 44±9 (media±ds) protein spots. All protein spots were excised from a preparative gel (200 µg), trypsin digested and analysed by MALDI-TOF-MS/MS: 24 proteins were identified. Conclusions: The microdialysis technique is a useful tool in the investigation of human metabolism. This is the first human adipose tissue proteomic characterization, 2-DE combined to mass spectrometry constitute a powerful tool to explore protein patterns of biological samples and differences in the microfiltrate proteome changes. The importance of identifying biomarkers of developing DM onset is given by the widespread dissemination of the same (90% of world population) considering that the disease is established very slowly and it takes a long time before symptoms can become clinically manifest. We have developed a protein extraction protocol and we obtained high-quality 2-DE map of human adipose tissue from which we have identified proteins. Dott.ssa Maria Grazia Nunno, phd c/o University of Foggia via Napoli, 52, Foggia Tel [email protected]

57 INDEX

58 L1 L2 L3 L4 L5 L6 1.O.1 PROTEINS PRODUCED BY PROBIOTIC BACTERIA AS MEDIATORS OF HOST-BACTERIA INTERACTIONS. A FOCUS ON BIFIDOBACTERIA. Dr. Abelardo Margolles PROTEOMICS DRIVEN TRANSLATIONAL RESEARCH IN PANCREATIC CANCER. Prof. Francesco Novelli AN "OMICS" APPROACH TO HUMAN LONGEVITY WITHIN A SYSTEMS BIOLOGY PERSPECTIVE. Prof. Claudio Franceschi PUSHING THE LIMITS OF PROTEOMICS TECHNIQUES TO UNDERSTAND STRESS AND TOLERANCE IN AN ALLOPOLYPLOID CROP Prof. Sebastien Carpentier PLANT GENOMES: INFORMATION CONTENT AS A START OF THE POSTGENOMIC ERA. Prof. Francesco Salamini NEXTPROT: THE HUMAN PROTEIN KNOWLEDGE PLATFORM IN THE CONTEXT OF THE HUMAN PROTEOME PROJECT Prof. Amos Bairoch FARM ANIMAL PROTEOMICS: A COST ACTION Paola Roncada 1.O.2 ANALYSIS OF THE SECRETOME OF THE GROUP B STREPTOCOCCUS Roberta Galbo, S. Papasergi, M. Garibaldi, I. Pernice, C. Lo Passo, F. Mandanici, L. Romeo, P. Trieu-Cuot, G. Teti and C. Beninati 1.O.3 2.O.1 2.O.2, 2.O.3 3.O.1 3.O.2 3.O.3 4.O.1 SOIL PROTEOMICS: INTERFERENCE OF SOIL SOLID PHASES ASSESSED BY MODEL STUDIES Laura Giagnoni, Andrea Migliaccio, Loretta Landi, Daniel van der Lelie, Safyh Taghavi, Paolo Nannipieri, Giancarlo Renella IN VITRO VITAMIN K TREATMENT IS NOT CAPABLE TO RESQUE UNEFFICIENT MGP- CARBOXYLATION IN PXE FIBROBLASTS Giulia Annovi, Federica Boraldi, Daniela Quaglino SEQUENTIAL APPLICATION OF URINARY PEPTIDE CLUSTERS OBTAINED AFTER WEAK- CATION EXCHANGE MAGNETIC BEAD PURIFICATION TO DETECT RENAL CELL CARCINOMA Erica Gianazza, Yuri E.M. van der Burgt, Italo Zoppis, Massimiliano Borsani, Giancarlo Mauri, Clizia Chinello, Valeria Squeo, Giancarlo Albo, Stefano Signorini, André M. Deelder, Marzia Galli Kienle and Fulvio Magni MODULATION OF URINARY PEPTIDOME IN HUMANS EXPOSED TO HIGH ALTITUDE HYPOXIA Veronica Mainini, Erica Gianazza, Clizia Chinello, Grzegorz Bilo, Miriam Revera, Andrea Giuliano, Gianluca Caldara, Carolina Lombardi, Alberto Piperno, Fulvio Magni, Gianfranco Parati EFFECTS OF AGEING IN MUSCLE TISSUE: THE CONTRIBUTION OF PROTEOMICS Daniele Capitanio, Michele Vasso, Cecilia Gelfi PROTEOMIC INVESTIGATIONS ON DIFFERENTIAL POST-TRANSLATIONAL MODIFICATIONS OF TRANSTHYRETIN IN MULTIPLE SCLEROSIS Damiana Pieragostinoa, Piero Del Boccioa, Maria Di Ioia, Sonia Buccia, Luisa Pieroni, Simona D Aguanno, Viviana Greco, Diego Centonze, Giovanna De Luca, Carmine Di Ilioa, Alessandra Lugaresia, Paolo Sacchetta, Andrea Urbania PROTEOMIC PROFILING OF BRAIN STEROID 5 ALPHA REDUCTASES IN SLEEP-DEPRIVATED RATS. Alessio Soggiu, Marco Bortolato, Paola Devoto, Cristian Piras, Luigi Bonizzi, Paola Roncada SIALYLATION DEFINING CELL INVASIVENESS IN BREAST CANCER Giuseppe Palmisano1, Rikke Leth-Larsen2, Martin Larsen1

59 4.O.2 4.O.3 PEPTIDOME PROFILING OF INDUCED SPUTUM BY MESOPOROUS SILICA BEADS AND MALDI-TOF MS FOR BIOMARKER DISCOVERY OF INFLAMMATORY LUNG DISEASES Rosa Terracciano, Mariaimmacolata Preianò, Grazia P. Palladino, Giovanna E. Carpagnano, Maria P. Foschino Barbaro, Girolamo Pelaia, Rosario Masellia and Rocco Savino GENOCOP ALGORITHM AND HIERARCHICAL GRID TRANSFORMATION FOR IMAGE WARPING OF TWO DIMENSIONAL GEL ELETROPHORETIC MAP Emilio Marengo, Elisa Robotti, Marco Demartini, Marina Cocchi, Daniela Cecconi 5.O.1 ARSENIC STRESS IN PTERIS VITTATA ROOTS COLONIZED BY ARBUSCULAR MYCORRHIZAL FUNGI Elisa Bona, Chiara Cattaneo, Patrizia Cesaro, Francesco Marsano, Graziella Berta and Maria Cavaletto 5.O.2 5.O.3 PROTEOMIC ANALYSIS OF A SPRING WHEAT CULTIVAR IN RESPONSE TO PROLONGED COLD STRESS Sara Rinalducci, Maria Giulia Egidi, Ghasem Karimzadeh, Ferdous Rastgar Jazii, Lello Zolla PROTEOMIC APPROACH TO CHARACTERIZE HOMOZYGOUS CITRUS CLEMENTINA HORT. EX TAN. GENOTYPES Maria Antonietta Germanà, Selenia Messina, Gianluca Di Cara, Nadia Ninfa Albanese, Alessia Pivetti, Maria Rita Marabeti, Ida Pucci-Minafra 1.1 PROTEOMIC AND FUNCTIONAL ANALYSIS OF HUMAN, BOVINE AND CAPRINE MILK FAT GLOBULES Stefano Spertino, Chiara De Angelis, Valentina Cipriani, Gabriella M. Giuffrida, Maria Cavaletto 1.2 MALDI-TOF MS PROTEOMIC PHENOTYPING BOOSTS PATIENT-TAILORED PROTOCOLS IN DIAGNOSTIC MYCOLOGY Federica Del Chierico, Andrea Masotti, Manuela Onori, Ersilia Fiscarelli, Leopoldo Dimiziani, Donato Menichella, Andrea Urbani and Lorenza Putignani 1.3 GROWTH-PHASE RELATED PROTEOMICS CAN ADD NEW INFORMATION TO THE DIFFERENT CATABOLIC ATTITUDE OF A. RADIORESISTENS S13 TOWARDS PHENOL AND BENZOATE. Paolo Fattori, Cristina Barello, Maria Gabriella Giuffrida, Carlo Giunta and Enrica Pessione 1.5 RND EFFLUX TRANSPORTER GENE DELETION IN BURKHOLDERIA CENOCEPACIA : A PROTEOMIC ANALYSIS Tania Gamberi, Maria Cristiana Papaleo, Silvia Rocchiccioli, Lorenzo Citti, Renato Fani, Alessandra Modesti 1.6 PROTEOMICS TO EVALUATE FERTILITY LEVEL OF BULLS SEMEN FROZEN WITH THREE DIFFERENT EXTENDERS Alessandro Gaviraghi, Alessio Soggiu, Andrea Galli, Cristian Piras, Pamela Bonaccini, Hany A. Hussein, Luigi Bonizzi, Paola Roncada 1.7 PROTEOMIC CHARACTERIZATION OF HUMAN MILK PROTEINS DURING A SIXTY-DAYS LACTATION PERIOD Alessandro Gaviraghi, Alessio Soggiu, Cristian Piras, Luigi Bonizzi, Carlo Agostoni, Paola Roncada 1.9 EXOPROTEOME OF THE PROBIOTIC LACTOBACILLUS REUTERI LB2 BM: WHAT HAPPENS IN THE HOST-PROKARIOTE INTERFACE Cristina Lamberti, Erika Mangiapane, Marco Cietto, Giuliana Lo Bianco, Ritva Virkola, Timo Korhonen, Enrica Pessione 1.10 A COMBINED ICP-MS AND CLASSICAL PROTEOMIC APPROACH TO ANALYZE SELENIUM UP-TAKE BY A PROBIOTIC LACTOBACILLUS REUTERI Erika Mangiapane, Eugenio Galano, Cristina Lamberti, Alessandro Pessione, Angela Amoresano, Pietro Pucci, Enrica Pessione 1.11 A PROTEOMIC APPROACH TO SEARCH BIOMARKERS USEFUL FOR IN VIVO SCRAPIE DIAGNOSIS Maria Mazza, Chiara Guglielmetti, Francesca Martucci, Marianna Pagano, Paola Marconi, Gianfranco Santagada, Pasquale Troiano, Maurizio Bruschi, Giovanni Candiano, Pier L. Acutis

60 1.12 IMMUNOGENIC PROTEINS AS PUTATIVE TARGETS FOR MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS DETECTION IN CATTLE Cristian Piras, Alessio Soggiu, Luigi Bonizzi, Gian Franco Greppi, Norma Arrigoni, Paola Roncada 1.13 PROTEOME ANALYSIS OF POST-MORTEM CHANGES IN BOVINE LONGISSIMUS DORSI BY TWO-DIMENSIONAL (2-DE), P-DIMENSIONAL (2-PE) ELECTROPHORESIS AND RANKING-PCA Michele Menini, Rita Polati, Elisa Robotti, Luca Fasolato, Filomena Montemurro, Emilio Marengo, Renato Millioni, and Daniela Cecconi 1.15 PROTEOMIC INVESTIGATION OF A NEW POSSIBLE OMEGA-OXIDANT BACTERIUM: A STUDY ON ACINETOBACTER RADIORESISTENS S13 Zapponi Michele, Fattori Paolo, Mazzoli Roberto, Pessione Alessandro, Giunta Carlo and Pessione Enrica 1.16 PVL RELATED DIFFERENCES IN PROTEIN EXPRESSION PROFILE IN MRSA AND MSSA Hany A. Hussein 1, Alessandro Gaviraghi 1, Cristian Piras 2, Alessio Soggiu 3, Monica Monaco 4, AnnalisaPantosti 4, Marco Tinelli 5, Luigi Bonizzi 1, Paola Roncada ANALYSIS OF ENDOTHELIAL CELL SECRETOME IDENTIFIED PENTRAXIN-3 AS A NOVEL TARGET OF THE LIPID-LOWERING HMG-COA REDUCTASE INHIBITORS Sabrina Lento, Maura Brioschi, Valentina Bozzola, Sabrina Galli, Elena Tremoli and Cristina Banfi 2.3 THE PROTEOME OF CARDIAC MICROPARTICLES Maura Brioschi, Simona Scarpella, Sabrina Galli, Elena Tremoli, Cristina Banfi 2.4 ERYTHROCYTES PRXII: OLIGOMERIC CHARACTERIZATION OF A CANDIDATE BIOMARKER OF OXIDATIVE STRESS DURING STORAGE UNDER BLOOD BANKING CONDITION. Barbara Blasi, Sara Rinalducci, Gian Maria D Amici, Lello Zoll 2.5 DIFFERENTIAL PROTEIN EXPRESSION PROFILING IN HUMAN TEARS OF PATIENTS WITH OPHTHALMOLOGIC DISEASES BY DIFFERENT PROTEOMIC APPROACHES Damiana Pieragostino, Sonia Bucci, Luca Agnifili, Vincenzo Fasanella, Simona D Aguanno, Marco Ciancaglini, Leonardo Mastropasqua, Andrea Urbani, Carmine Di Ilio, Paolo Sacchetta and Piero Del Boccio 2.6 MOUSE TO HUMAN AUTOANTIBODY SIGNATURE IN PANCREATIC DUCTAL ADENOCARCINOMA Michela Capello, Paola Cappello, Federica C. Linty, Sammy Ferri-Borgogno, Roberto Chiarle, Aldo Scarpa, Paolo Pederzoli, Paola Nisticò, Michele Milella and Francesco Novelli 2.7 AN ALTERNATIVE APPROACH FOR THE IDENTIFICATION AND QUANTIFICATION OF POTENTIAL PROSTATE CANCER BIOMARKERS. Francesca Casadonte, Anna Napoli, Donatella Aiello, Rocco Damiano, Rosa Terracciano, Giovanni Sindona and Rocco Savino 2.8 HUMAN SALIVARY PROTEOME AND BITTER TASTE: THE RECIPROCAL INFLUENCE Tiziana Cabras, Melania Melis, Alessandra Padiglia, Massimo Castagnola, Irene Messana, Iole Tomassini Barbarossa 2.9 CAN PROTEOMIC APPROACH HELP US IN DIAGNOSIS OF RIEDEL S THYROIDITIS? Federica Ciregia, Pietro Iacconi, Laura Giusti, Ylenia Da Valle, Elena Donadio, Gino Giannaccini, Agnese Proietti, Liborio Torregrossa, Antonio Lucacchini 2.10 COORDINATED CHANGES OF ADHESION ELEMENTS MAY REGULATE PHENOTYPE SWITCH IN VASCULAR SMOOTH MUSCLE CELLS Silvia Rocchiccioli, Antonella Cecchettini, Nadia Ucciferri, Maria Giovanna Trivella, Lorenzo Citti 2.11 IDENTIFICATION OF NUCLEAR SUBSTRATES OF AKT/PKB BY FUNCTIONAL PROTEOMICS: PROHIBITIN 2 IS A TARGET OF AKT PHOSPHORYLATION IN HUMAN PROMYELOCYTIC LEUKEMIA CELLS. Antonietta D Angelo, Alberto Bavelloni, Manuela Piazzi, William Blalock, Francesca Tagliavini, Irene Faenza, Diego Pinetti, Lucio Cocco

61 2.12 PROTEOMIC PROFILE OF WASHING FLUID OF COLORECTAL TRACT TO SEARCH POTENTIAL BIOMARKERS OF COLON CANCER Ylenia Da Valle, Pietro Iacconi, Federica Ciregia, Laura Giusti, Tiziana Ventroni, Elena Donadio, Gino Giannaccini, Massimo Chiarugi, Liborio Torregrossa, Agnese Proietti, Fulvio Basolo, Antonio Lucacchini 2.13 LIPIDOMIC APPROACH ON URINARY EXOSOMES FROM RCC PATIENTS: AN INSIGHT INTO THE TUMOR BIO-SIGNATURE Piero Del Boccio, Francesca Raimondo, Damiana Pieragostino, Lavinia Morosi, Francesco Rocco, Paolo Brambilla, Claudia Augello, Andrea Urbani, Paolo Sacchetta, Fulvio Magni, Marina Pitto 2.14 ANALYSIS OF TRASTUZUMAB EFFECTS IN BREAST CANCER CELLS IN VITRO Gianluca Di Cara, Nadia N. Albanese, Rosa Musso, Francesca Costantini, Maria R. Marabeti, Patrizia Cancemi, Ida Pucci-Minafra 2.15 PROTEIN CARBONYLATION IN PERIPHERAL BLOOD MONONUCLEAR CELLS FROM AMYOTROPHIC LATERAL SCLEROSIS PATIENTS Assunta Gagliardi, Simona Frosali, Claudia Landi, Laura Bianchi, Maria Cipriano, Michele Puglia, Anna Gimigliano, Alessandro Armini, Anna Di Stefano, Luca Bini 2.17 A PROTEOMIC STUDY OF HEPATOCELLULAR CARCINOMA DISEASE: A COMPARISON BETWEEN THE LIVER INFLAMMATORY CONDITION AND CANCER OUTCOME BY DIGE APPROACH Gimigliano Anna, Martins M.Aline, Bianchi Laura, Puglia Michele,Landi Claudia a Gagliardi Assunta, Cipriano Maria, Armini Alessandro, Bini Luca DIGE ANALYSIS OF OVARIAN CANCER CELL RESPONSES TO CYTOTOXIC GOLD COMPOUNDS AND ANTIMETASTATIC RUTHENIUM COMPOUNDS Francesca Guidi, Michele Puglia, Francesca Magherini, Luca Bini, Enrico Mini, Chiara Gabbiani, Luigi Messori and Alessandra Modesti 2.19 PROTEOMIC ANALYSIS OF INTERSTITIAL LUNG DISEASES Landi Claudia, Bargagli Elena, Perari Maria Grazia, Puglia Michele, Gagliardi Assunta, Cipriano Maria, Bianchi Laura, Gimigliano Anna, Armini Alessandro, Rottoli Paola, Bini Luca 2.20 PROTEOMIC ANALYSIS OF HUMAN GLIOBLASTOMA CELL LINES WITH DIFFERENT RESPONSE TO NITRIC OXIDE. Roberta Leone, Paola Giussani, Agnese Viganò, Michele Vasso, Daniele Capitanio, Chiara Fania, Paola Viani, Cecilia Gelfi 2.21 MALDI IMAGING: A NEW TOOL FOR MAPPING MULTIPLE SCLEROSIS BRAIN LESIONS TO LOOK BEYOND CLASSICAL HISTOLOGY Giuseppina Maccarrone, Sandra Nischwitz, Søren-Oliver Deininger, Joachim Hornung, Fatima König, Christine Stadelmann-Nessler, Frank Weber, Chris W. Turck 2.23 TIME-COURSE INVESTIGATION OF SAGM-STORED ERYTHROCYTE CONCENTRATES: FROM METABOLISM TO PROTEOMICS Cristina Marrocco, Gian M. D Amici, Angelo D Alessandro, Valeria Pallotta, Sara Rinalducci and Lello Zolla 2.24 PROTEOMIC AND MORPHOLOGICAL CHARACTERIZATION OF RCC URINARY EXOSOMES Lavinia Morosi, Francesca Raimondo, Pamela Della Mina, Antonello Villa, Clizia Chinello, G Cozzi, Paolo Brambilla, Fulvio Magni a and Marina Pitto 2.25 DIFFERENTIAL PROTEOMIC ANALYSIS OF THYROID CARCINOMA CELL LINES Rosa Musso, Gianluca Di Cara, Nadia N. Albanese, Francesca Costantini, Alessia Pivetti, Maria R. Marabeti, Giovanni Zito, Patrizia Cancemi, Carla Giordano and Ida Pucci-Minafra 2.27 HEART PROTEOMICS OF GUINEA PIGS Gianluca Paredi, Luca Ronda, Stefano Bruno, Simona Bertoni, Elisabetta Barocelli, and Andrea Mozzarelli

62 2.28 DOPAMINE RECEPTOR AGONISTS AND LEVODOPA MODULATE PROTEIN LEVELS IN HUMAN T LYMPHOCYTES. Agnese C. Pippione, Tiziana Alberio, Daniela Cecconi, Simone Olgiati, Maurizio Zibetti, Leonardo Lopiano, Mauro Fasano 2.29 PROTEOMICS OF RCC MEMBRANE MICRODOMAINS Raimondo Francesca, Morosi Lavinia, Mainini Veronica, Bianchi Cristina, Albo Giancarlo, Ferrero Stefano, Magni Fulvio a and Pitto Marina 2.30 CREUTZFELDT-JAKOB DISEASE AND OTHER DEMENTIA MAY BE DISCRIMINATED BY THYMOSIN BETA 4 LEVELS IN CEREBROSPINAL FLUID USING MALDI-TOF MS. Elena Urso, Maria Le Pera, Sabrina Bossio, Teresa Sprovieri, Ida Manna, Chiara Cupidi, Umberto Aguglia, Antonio Gambardella, Aldo Quattrone, Antonio Qualtieri 2.31 PROTEOMIC PROFILE OF CANCER STEM CELLS DIFFERENTIATION SHOWS ALTERATION OF GLYCOLYTIC PATHWAY Domenico Ciavardelli, Claudia Rossi, Federica Forlì, Daniela Barcaroli, Mirco Zucchelli, Simona D Aguanno, Paolo Sacchetta, Carmine Di Ilio, Matilde Todaro, Giorgio Stassi, Vincenzo De Laurenzi and Andrea Urbani 2.32 VALIDATION OF A BIOMARKER PANEL FOR RCC Claudia Salemi, Daniela Fumagalli, Francesca Raimondo, Clizia Chinello, Francesco Rocco, Stefano Ferrero, Cecilia Sarto, Marina Pitto, Paolo Brambilla and Fulvio Magni 2.33 LYMPHOBLASTOID PROTEOME STUDY IN BIPOLAR DISORDER PATIENTS TREATED WITH LITIUM. Alessio Soggiu, Cristian Piras, Alessio Squassina, Maria Del Zompo, Luigi Bonizzi, Paola Roncada 2.34 PHYTOCHEMICALS RESVERATROL AND CURCUMIN AS POTENTIAL AGENTS IN THE PREVENTION AND THERAPY OF OVARIAN AND BREAST CANCERS Daniele Vergara, Daniela Toraldo, Piero del Boccio, Damiana Pieragostino, Pasquale Simeone, Andrea Tinelli, Stefania De Domenico, Raffaele Acierno, Giovanna Giovinazzo, Andrea Urbani, Angelo Santino, Michele Maffia 2.35 EFFECTS OF DEGENERATION-REGENERATION INDUCED BY NERVE CRUSH IN RAT GASTROCNEMIUS MUSCLE. Laura Barbalini 1, Daniele Capitanio 1, Chiara Fania 1, Michele Vasso 1,2, Roberta Leone 1, Isabelle Perroteau 3, Stefano Geuna 4, Cecilia Gelfi 1,2 3.2 PROTEOMICS TO EVALUATE THE EFFECT OF CRYOPRESERVATION ON POST-THAW STALLION SPERM Pamela Bonaccini, Alessandro Gaviraghi, Hani Husssein, Luigi Bonizzi, Stefano Romagnoli, MariaElena Falomo, Paola Roncada 3.3 OVOTRANSFERRIN AND GALLINACIN-9 FOUND IN SEMINAL PLASMA OF ROOSTER Annelisse Castillo, Margherita Marzoni, Simona Sagona, Lorenzo Citti, Silvia Rocchiccioli, Isabella Romboli, Antonio Felicioli 3.4 IL-8 TRANSCRIPTIONAL MACHINERY IN RESPIRATORY EPITHELIAL CELLS EXPOSED TO PRO-INFLAMMATORY STIMULI SPECIFIC OF CF LUNG DISEASE Carla Iannone, Maria Monti, Valentino Bezzeri, Monica Borgatti, Roberto Gambari, Giulio Cabrini and Piero Pucci 3.7 DESIGN OF A BIOINFORMATICS ALGORITHM FOR THE ASSESSMENT OF MOUSE GUT PHYLOTYPES IDENTIFIED BY METAPROTEOMIC APPROACH Lorenza Putignani, Stefano Levi Mortera, Federica Del Chierico, Pamela Vernocchi, M. Manuela Rosado, Rita Carsetti, and Andrea Urbani 4.1 COMBINING TOP DOWN AND BOTTOM UP ANALYSES IN A SINGLE LCMS EXPERIMENT Reinaldo Almeida

63 4.2 TOP-DOWN RP-HPLC-ESI-MS PLATFORMS FOR THE DETECTION OF THE INTACT NATURALLY OCCURRING HUMAN SALIVARY PROTEOME. Massimo Castagnola, Federica Iavarone, Chiara Fanali, Rosanna Inzitari, Federica Vincenzoni, Tiziana Cabras, Barbara Manconi, Maria T. Sanna, Alessandra Olianas, Elisabetta Pisano, Roberto Boi, Sonia Nemolato, Alberto Vitali, Emanuele Scarano, Antonella Fiorita, Giulio C. Passali, Armando Manni, Massimo Cordaro,Gaetano Paludetti, Gavino Faa, Irene Messana 4.3 THE ORBITRAP TECHNOLOGY: INCREASED ANALYTICAL PERFORMANCES FOR THE ANALYSIS OF PEPTIDES AND PROTEINS. Martin Zeller; Catharina Crone; Carmen Paschke; Mathias Mueller; Eugen Damoc; Eduard Denisov; Alexander Makarov; Dirk Nolting; Bernard Dealnghe; Thomas Moehring 4.7 TARGETED TOP-DOWN PROTEOMIC ANALYSIS OF CEREBROSPINAL FLUID FROM AMYOTROPHIC LATERAL SCLEROSIS PATIENTS Marta Vilaseca, Claudio Diema, Núria Omeñaca, Alex Campos, Eliandre de Oliveira, Mark Baumert, Kees Vlak, Joan Guinovart, Jacques Borg 4.8 ULTRAHIGH-PERFORMANCE NANO LC-MS/MS ANALYSIS OF COMPLEX PROTEOMIC SAMPLES Steffen Liedtke Evert-Jan Sneekes Bjorn de Haan and Remco Swart 4.9 TWO DIMENSIONAL SEC // RP CAPILLARY LC FOR TOP-DOWN PROTEOMICS ANALYSIS Robert van Ling, Franco Abballe, Evert-Jan Sneekes, Perry van Paassen, Macro Karsten, Wim Decrop, Remco Swart 4.10 OPTIMIZING PARTICLE SIZE AND COLUMN LENGTH, WHAT IS THE BEST WAY TO UTILIZE NANO UHPLC IN PROTEOMICS? Robert van Ling, Franco Abballe, Evert-Jan Sneekes, Macro Karsten, Wim Decrop, Remco Swart 4.11 EXPLORATION OF PH-GRADIENT, ION-EXCHANGE CHROMATOGRAPHY FOR HIGH- RESOLUTION PROTEIN SEPARATIONS IN BIOTECHNOLOGY AND PROTEOMICS Wim Decrop, Marie-Jeanne Olivo, Evert-Jan Sneekes, and Remco Swar 4.12 SET-UP FOR HUMAN SERA MALDI PROFILING: THE CASE OF RHEPO TREATMENT Enrica Torretta 1, Chiara Fania 1, Michele Vasso 1,2, Carsten Lundby 3, Cecilia Gelfi 1,2 5.2 CHARACTERIZATION OF ANTENNA ISOFORMS FROM SPINACH LEAVES GROWN UNDER DIFFERENT LIGHT CONDITIONS Federica Gevi, Anna Maria Timperio, Sara Rinalducci, Maria Giulia Egidi, Lello Zolla 5.5 SALT STRESS RESPONSES IN PINUS HALEPENSIS Lomaglio Toni, Satriani, A, Verrillo, F, Rocco, M. 5.6 PROTEOMICS OF THE RIPENING FRUIT IN OLEA EUROPAEA Linda Bianco, Luciana Baldoni, Fiammetta Alagna, Christine Finnie, Birte Svensson, Silvia Mazzuca and Gaetano Perrotta

64

65 NAME INDEX

66 Abballe F. 4.9, 4.10 Acierno R Acutis P.L Agnifili L. 2.5 Agostini C. 1.7 Aguglia U Aiello D. 2.7 Alagna F. 5.6 Albanese N.N. 5.O.3, 2.14, 2.25 Alberio T. 2.28, Albo G. 2.O.2, 2.29, Almeida R. 4.1, Amoresano A. 1.10, Annovi Giulia 2.O.1, Armini A. 2.15, 2.17, 2.19, Arrigoni N. 1.12, Augello C. 2.13, Bairoch A. L6 Baldoni L. 5.6, Banfi C. 2.3, Barbalini L Barcaroli D. 2.31, Barello C. 1.3, Bargagli E. 2.19, Barocelli E. 2.27, Basolo F. 2.12, Baumert M. 4.7, Bavelloni A. 2.11, Beninati C. 1.O.2, Berta P. 5.O.1, Bertoni S. 2.27, Bezzeri V. 3.4, Bianchi C. 2.29, Bianchi L. 2.15, 2.17, 2.19, Bianco L. 5.6, Bilo G. 2.O.3

67 Bini L. 2.15, 2.17, 2.18, 2.19, Bucci S. 3.O.2, 2.5, Blalock W. 2.11, Blasi B. 2.4, Boi R. 4.2, Bona Elisa 5.O.1, Bonaccini P. 1.6, 3.2, Bonizzi L. 3.O.3, 1.6, 1.7, 1.12, 1.16, 2.33, 3.2, Boraldi F. 2.O.1, Borg J. 4.7, Borgatti M. 3.4, Borsani M. 2.O.2, Bortolato M. 3.O.3, Bossio S. 2.30, Bozzola V. 2.2, Brambilla P. 2.13, 2.24, 2.32, Brioschi M. 2.2, Bruno S. 2.27, Bruschi M. 1.11, Cabras T. 2.8, 4.2, Cabrini G. 3.4, Caldara G. 2.O.3 Campos A. 4.7, Cancemi P. 2.14, 2.25, Candiano G. 1.11, Capello M. 2.6, Capitanio D. 3.O.1, 2.20, 2.35 Cappello P. 2.6, Carpagnano G.E. 4.O.2, Carpentier S. L4 Carsetti R. 3.7, Casadonte F. 2.7, Castagnola M. 2.8, 4.2, Castillo A. 3.3, Cattaneo C. 5.O.1, Cavaletto Maria 5.O.1, 1.1,

68 Cecchettini A. 2.10, Cozzi G. 2.24, Cecconi D. 4.O.3, 1.13, 2.28, Centonze D. 3.O.2 Cesaro P. 5.O.1, Chiarle R. 2.6, Chiarugi M. 2.12, Chinello C. 2.O.2, 2.O.3, 2.24, 2.32, Ciancaglini M. 2.5, Ciavardelli D. 2.31, Cietto M. 1.9, Cipriani V. 1.1, Cipriano M. 2.15, 2.17, 2.19, Ciregia F. 2.9, 2.12, Citti L. 1.5, 2.10, 3.3, Cocchi M. 4.O.3, Cocco L. 2.11, Cordaro M. 4.2, Costantini F. 2.14, 2.25, Crone C. 4.3, Cupidi C. 2.30, D'Aguanno S. 3.O.2, 2.5, 2.31, D'Alessandro A. 2.23, D'Amici G.M. 2.4, 2.23, D'Angelo A. 2.11, Da Valle Y. 2.9, 2.12, Damiano R. 2.7, Damoc E. 4.3, De Angelis C. 1.1, De Domenico S. 2.34, De Laurenzi V. 2.31, De Luca G. 3.O.2 De Stassi G. 2.31, Dealnghe B. 4.3, Decrop W. 4.9, 4.10, 4.11, Deelder A. 2.O.2,

69 Deininger S-O. 2.21, Falomo M.E. 3.2, Del Boccio P. 3.O.2, 2.5, 2.13, 2.34 Del Chierico F. 1.2, 3.7, Del Zompo M. 2.33, Della Mina P. 2.24, Demartini M. 4.O.3, Denisov E. 4.3, Devoto P. 3.O.3, Di Cara G. 5.O.3, 2.14, 2.25, Di Ilio C. 3.O.2, 2.5, 2.31, Di Ioia M. 3.O.2 Di Stefano A. 2.15, Diema C. 4.7, Dimiziani L. 1.2, Donadio E. 2.9, 2.12, Egidi M.G. 5.O.2, 5.2, Faa G. 4.2, Faenza I. 2.11, Fanali C. 4.2, Fani R. 1.5, Fania C. 2.20, 2.35, 4.12 Fasanella V. 2.5, Fasano M. 2.28, Fasolato L. 1.13, Fattori P. 1.3, 1.15, Felicioli A. 3.3, Ferrero S. 2.29, 2.32, Ferri-Borgogno S. 2.6, Finnie C. 5.6, Fiorita A. 4.2, Fiscarelli E. 1.2, Forlì F. 2.31, Foschino Barbaro M.P. 4.O.2, Franceschi Claudio L3 Frosali S. 2.15,

70 Fumagalli D. 2.32, Gabbiani C. 2.18, Gagliardi A. 2.15, 2.17, 2.19, Galano E. 1.10, Galbo Roberta 1.O.2, Galli A. 1.6, Galli Kienle M. 2.O.2, Galli S. 2.2, Gambardella A. 2.30, Gambari R. 3.4, Gamberi Tania 1.5, Garibaldi M. 1.O.2, Gaviraghi A. 1.6, 1.7, 1.16, 3.2, Gelfi C. 3.O.1, 2.20, 2.35, 4.12 Germanà M.A. 5.O.3, Geuna S Gevi F. 5.2, Giagnoni Laura 1.O.3, Gianazza E. 2.O.2, 2.O.3, Giannaccini G. 2.9, 2.12, Gimigliano A. 2.15, 2.17, 2.19, Giordano C. 2.25, Giovinazzo G. 2.34, Giuffrida M.G. 1.1, 1.3, Giuliano A. 2.O.3 Giunta C. 1.3, 1.15, Giussani P. 2.20, Giusti L. 2.9, 2.12, Greco V. 3.O.2 Greppi G.F. 1.12, Guglielmetti C. 1.11, Guidi F. 2.18, Guinovart J. 4.7, Haan de B. 4.8, Hornung J. 2.21, Hussein H.A. 1.6, 1.16, 3.2,

71 Iacconi P 2.9, 2.12, Iannone C. 3.4, Ling van R. 4.9, 4.10, Linty F.C. 2.6, Iavarone F. 4.2, Inzitari R. 4.2, Karimzadeh G. 5.O.2, Karsten M. 4.9, Konig F. 2.21, Korhonen T. 1.9, Lamberti C. 1.9, 1.10, Landi C. 2.15, 2.17, 2.19, Landi L. 1.O.3, Larsen M. 4.O.1, Le Pera M. 2.30, Lento S. 2.2, Leone R. 2.10, 2.35 Leth-Larsen R. 4.O.1, Levi Mortera S. 3.7, Liedtke S. 4.8, Lo Bianco G. 1.9, Lo Passo C. 1.O.2, Lomaglio T. 5.5, Lombardi C. 2.O.3 Lopiano L. 2.28, Lucacchini A. 2.9, 2.12, Lugaresi A. 3.O.2 Lundby C Maccarrone G. 2.21, Maffia M. 2.34, Magherini F. 2.18, Magni F. 2.O.2, 2.O.3, 2.13, 2.24, 2.29, 2.32, Mainini V. 2.O.3, 2.29, Makarov A. 4.3, Manconi B. 4.2, Mandanici F. 1.O.2,

72 Mangiapane E. 1.9, 1.10, Manna I. 2.30, Manni A. 4.2, Mazzucca S. 5.6, Melis M. 2.8, Menichella D. 1.2, Marabeti M.R. 5.O.3, 2.14, 2.25, Marconi P. 1.11, Marengo E. 4.O.3, 1.13, Margolles Abelardo L1 Marocco C. 2.23, Marsano F. 5.O.1, Martins M.A. 2.17, Martucci F. 1.11, Marzoni M. 3.3, Maselli R. 4.O.2, Masotti A. 1.2, Mastropasqua L. 2.5, Mauri G. 2.O.2, Mazza M. 1.11, Mazzoli R. 1.15, Menini M. 1.13, Messana I. 2.8, 4.2, Messina S. 5.O.3, Messori L. 2.18, Migliaccio A. 1.O.3, Milella M. 2.6, Millioni R. 1.13, Mini E. 2.18, Modesti A. 1.5, 2.18, Moehring T. 4.3, Monaco M Montemurro F Monti M. 3.4, Morosi L. 2.13, 2.24, 2.29, Mozzarelli A. 2.27,

73 Mueller M. 4.3, Musso R. 2.14, 2.25, Nannipieri P. 1.O.3, Napoli A. 2.7, Nemolato S. 4.2, Nischwitz S. 2.21, Nistico P. 2.6, Nolting D. 4.3, Novelli F. L2, 2.6, Olgiati S. 2.28, Olianas A. 4.2, Oliveira de E. 4.7, Olivo M-J Omenaca N. 4.7, Onori M. 1.2, Paassen van P. 4.9, 4.10, Padiglia A. 2.8, Pagano M. 1.11, Palladino G.P. 4.O.2, Pallotta V. 2.23, Palmisano Giuseppe 4.O.1, Paludetti G. 4.2, Pantosti A Papaleo M.C. 1.5, Papasergi S. 1.O.2, Parati G. 2.O.3 Paredi G. 2.27, Paschke C. 4.3, Passali G.C. 4.2, Pederzoli P. 2.6, Pelaia G. 4.O.2, Perari M.G. 2.19, Pernice I. 1.O.2, Perrotta G. 5.6, Perroteau I Pessione A. 1.10, 1.15, Pessione E.

74 1.3, 1.9, 1.10, 1.15, 2.O.1, Piazzi M. 2.11, Pieragostino D. 3.O.2, 2.5, 2.13, 2.34, Pieroni L. 3.O.2 Pinetti D, 2.11, Piperno A. 2.O.3 Pippione A.C. 2.28, Piras C. 3.O.3, 1.6, 1.7, 1.12, 1.16, 2.33, Pisano E. 4.2, Pitto M. 2.13, 2.24, 2.29, 2.32, Pivetti A. 5.O.3, 2.25, Polati R. 1.13, Preianò M. 4.O.2, Proietti A. 2.9, 2.12, Pucci P. 1.10, 3.4, Pucci-Minafra I. 5.O.3, 2.14, 2.25, Puglia M. 2.15, 2.17, 2.18, 2.19, Putignani L. 1.2, 3.7, Quaglino D. Qualtieri A. 2.30, Quattrone A. 2.30, Raimondo F. 2.13, 2.24, 2.29, 2.32, Rastgar Jazii F. 5.O.2, Renella G. 1.O.3, Revera M. 2.O.3 Rinalducci S. 5.O.2, 2.4, 2.23, 5.2, Robotti E. 4.O.3, 1.13, Rocchiccioli S. 1.5, 2.10, 3.3, Rocco F. 2.13, 2.32, Rocco M. 5.5, Romagnoli S. 3.2, Romboli I. 3.3, Romeo L. 1.O.2, Roncada P. 1.O.1, 3.O.3, 1.6, 1.7, 1.12, 1.16, 2.33, 3.2, Ronda L. 2.27, Rosado M. 3.7,

75 Rossi C. 2.31, Rottoli P. 2.19, Sacchetta P. 3.O.2, 2.5, 2.13, 2.31, Sagona S. 3.3, Salamini F. L5 Salemi C. 2.32, Soggiu A. 3.O.3, 1.6, 1.7, 1.12, 1.16, 2.33, Spertino S. 1.1, Sprovieri T. 2.30, Squassina A. 2.33, Squeo V. 2.O.2, Stadelmann-Nessler C. 2.21, Sanna M.T. 4.2, Santagada G. 1.11, Santino A. 2.34, Sarto C. 2.32, Satriani A. 5.5, Savino R. 4.O.2, 2.7, Scarano E. 4.2, Scarpa A. 2.6, Signorini S. 2.O.2, Simeone P. 2.34, Sindona G. 2.7, Sneekes E-J. 4.8, 4.9, 4.10, 4.11, Stassi G. 2.31, Svensson B. 5.6, Swart R. 4.8, 4.9, 4.10, 4.11, Taghavi S. 1.O.3, Tagliavini F. 2.11, Terracciano R. 4.O.2, 2.7, Teti G. 1.O.2, Timperio A.M. 5.2, Tinelli A. 2.34, Tinelli M Todaro M. 2.31, Tomassini Barbarossa I. 2.8,

76 Toraldo D. 2.34, Torregrossa L. 2.9, 2.12, Torretta E Tremoli E. 2.2, Trieu-Cuot P. 1.O.2, Trivella M.G. 2.10, Troiano P. 1.11, Turck C.W. 2.21, Ucciferri N. 2.10, Urbani A. 3.O.2, 1.2, 2.5, 2.13, 2.31, 2.34, 3.7, Urso E. 2.30, Van der Burgt Y. 2.O.2, Van der Lelie D. 1.O.3, Vasso M. 3.O.1, 2.20, 2.35, 4.12 Ventroni T. 2.12, Vergara D. 2.34, Verrillo F. 5.5, Viani P. 2.20, Viganò A. 2.20, Vilaseca M. 4.7, Villa A. 2.24, Vincenzoni F. 4.2, Virkola R. 1.9, Vitali A. 4.2, Vlak K. 4.7, Weber F. 2.21, Zapponi M. 1.15, Zeller M. 4.3, Zibetti M. 2.28, Zito G. 2.25, Zolla L. 5.O.2, 2.4, 2.23, 5.2, Zoppis I. 2.O.2, Zucchelli M. 2.31, Vernocchi P. 3.7,

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