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‘One Health’ summarises the idea that human health and animal health are interdependent and bound to the health of ecosystems. The purpose of proteomics methodologies and studies is to determine proteins present in samples of interest and to quantify changes in protein expression during pathological conditions. The objectives of this paper are to review the application of proteomics technologies within the One Health concept and to appraise their role in the elucidation of diseases and situations relevant to One Health. The paper develops in three sections. Proteomics Applications in Zoonotic Infections part discusses proteomics applications in zoonotic infections and explores the use of proteomics for studying pathogenetic pathways, transmission dynamics, diagnostic biomarkers and novel vaccines in prion, viral, bacterial, protozoan and metazoan zoonotic infections. Proteomics Applications in Antibiotic Resistance part discusses proteomics applications in mechanisms of resistance development and discovery of novel treatments for antibiotic resistance. Proteomics Applications in Food Safety part discusses the detection of allergens, exposure of adulteration, identification of pathogens and toxins, study of product traits and characterisation of proteins in food safety. Sensitive analysis of proteins, including low-abundant ones in complex biological samples, will be achieved in the future, thus enabling implementation of targeted proteomics in clinical settings, shedding light on biomarker research and promoting the One Health concept.

The national infrastructure FoodOmicsGR_RI coordinates research efforts from eight Greek Universities and Research Centers in a network aiming to support research and development (R&D) in the agri-food sector. The goals of FoodOmicsGR_RI are the comprehensive in-depth characterization of foods using cutting-edge omics technologies and the support of dietary/nutrition studies. The network combines strong omics expertise with expert field/application scientists (food/nutrition sciences, plant protection/plant growth, animal husbandry, apiculture and 10 other fields). Human resources involve more than 60 staff scientists and more than 30 recruits. State-of-the-art technologies and instrumentation is available for the comprehensive mapping of the food composition and available genetic resources, the assessment of the distinct value of foods, and the effect of nutritional intervention on the metabolic profile of biological samples of consumers and animal models. The consortium has the know-how and expertise that covers the breadth of the Greek agri-food sector. Metabolomics teams have developed and implemented a variety of methods for profiling and quantitative analysis. The implementation plan includes the following research axes: development of a detailed database of Greek food constituents; exploitation of “omics” technologies to assess domestic agricultural biodiversity aiding authenticity-traceability control/certification of geographical/genetic origin; highlighting unique characteristics of Greek products with an emphasis on quality, sustainability and food safety; assessment of diet’s effect on health and well-being; creating added value from agri-food waste. FoodOmicsGR_RI develops new tools to evaluate the nutritional value of Greek foods, study the role of traditional foods and Greek functional foods in the prevention of chronic diseases and support health claims of Greek traditional products. FoodOmicsGR_RI provides access to state-of-the-art facilities, unique, well-characterised sample sets, obtained from precision/experimental farming/breeding (milk, honey, meat, olive oil and so forth) along with more than 20 complementary scientific disciplines. FoodOmicsGR_RI is open for collaboration with national and international stakeholders.

Background/aim Proteomics technologies provide fundamental insights into the high organizational complexity and diversity of the central nervous system. In the present study, high-resolution mass spectrometry (MS) was applied in order to identify whole-proteome content of anatomically distinct and functionally specific mouse brain regions. Materials and methods Brains from eight 8-week-old C57BL/6N normal male mice were separated into seven anatomically district regions. The protein content of each region was analyzed by high-throughput nano-liquid chromatography-MS/MS Orbitrap elite technology. Results A total of 16,574 proteins were identified: 2,795 in cerebral cortex, 2,311 in olfactory bulb, 2,246 in hippocampus, 2,247 in hypothalamus, 2,250 in mid brain, 2,334 in cerebellum and 2,391 in medulla. Of these proteins, 534 were uniquely expressed in cerebral cortex, 323 in olfactory bulb, 230 in hippocampus, 272 in hypothalamus, 1,326 in mid brain, 320 in cerebellum and 268 in medulla. Conclusion These data represent the most comprehensive proteomic map of the normal mouse brain and they might further be used in studies related to brain diseases, including cancer and neurodegenerative diseases.

Background To date, the elucidation of serum protein alterations in male breast cancer (MBC) has not been extensively studied, due to the rarity of the disease. Materials and methods In the present work, two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) were employed to detect differences in serum protein expression between patients with MBC and healthy controls. Results A panel of differentially expressed serum proteins was identified, including proteins involved in the regulation of the cell cycle [e.g. cell division cycle 7-related protein kinase (CDC7)], in mitochondrial function [e.g. mitochondrial aldehyde dehydrogenase (ALDH2) and dimethyladenosine transferase 1 (TFB1M)], in lipid metabolism and transport [e.g. apolipoprotein A-I (APOA1) and E (APOE)], in apoptosis and immune response [e.g. CD5 antigen-like (CD5L), clusterin (CLUS) and C-C motif chemokine 14 (CCL14)], in transcription (e.g. protein SSX3 and signal transducer and activator of transcription 3 (STAT3)], in invasion and metastasis (e.g. alpha-2-HS-glycoprotein (FETUA)], in estrogen synthesis [aromatase (CYP19A1)] and other diverse biological roles [e.g. actin-related protein 2/3 complex subunit 4 (ARPC4), dual specificity mitogen-activated protein kinase kinase 4 (MP2K4), ectoderm-neural cortex protein 1 (ENC1), and matrix metalloproteinase-27 (MMP27)]. Conclusion These findings provide valuable insight into the distinct clinicopathological features of MBC and indicate that select serum proteomic markers may help improve MBC management.

The global incidence of metabolic disorders like type 2 diabetes mellitus (DM2) has assumed epidemic proportions, leading to adverse health and socio-economic impacts. It is therefore of critical importance the early diagnosis of DM2 patients and the detection of those at increased risk of disease. In this respect, Precision Medicine (PM) is an emerging approach that includes practices, tests, decisions and treatments adapted to the characteristics of each patient. With regard to DM2, PM manages a wealth of “omics” data (genomic, metabolic, proteomic, environmental, clinical and paraclinical) to increase the number of clinically validated biomarkers in order to identify patients in early stage even before the prediabetic phase. Significance In this paper, we discuss the epidemic dimension of metabolic disorders like type 2 diabetes mellitus (DM2) and the urgent demand for novel biomarkers to reduce the incidence or even delay the onset of DM2. Recent research data produced by “multi-omics” technologies (genomics/epigenomics, transcriptomics, proteomics and metabolomics), suggest that many potential biomarkers might be helpful in the prediction and early diagnosis of DM2. Predictive, Preventive and Personalized Medicine (PPPM) manages and integrates these data to apply personalized, preventive, and therapeutic approaches. This is significant because there is an emerging need for establishing channels for communication and personalized consultation between systems research and precision medicine, as the medicine of the future.

The principles of Predictive, Preventive and Personalized Medicine (PPPM) dictate the need to recognize individual susceptibility to disease in a timely fashion and to offer targeted preventive interventions and treatments. Proteomics is a state-of-the art technology- driven science aiming at expanding our understanding of the pathophysiologic mechanisms that underlie disease, but also at identifying accurate predictive, diagnostic and therapeutic biomarkers, that will eventually promote the implementation of PPPM. In this review, we summarize the wide spectrum of the applications of Mass Spectrometry-based proteomics in the various fields of Pediatric Endocrinology, including Inborn Errors of Metabolism, type 1 diabetes, Adrenal Disease, Metabolic Syndrome and Thyroid disease, ranging from neonatal screening to early recognition of specific at-risk populations for disease manifestations or complications in adult life and to monitoring of disease progression and response to treatment. Significance Proteomics is a state-of-the art technology- driven science aiming at expanding our understanding of the pathophysiologic mechanisms that underlie disease, but also at identifying accurate predictive, diagnostic and therapeutic biomarkers that will eventually lead to successful, targeted, patient-centric, individualized approach of each patient, as dictated by the principles of Predictive, Preventive and Personalized Medicine. In this review, we summarize the wide spectrum of the applications of Mass Spectrometry-based proteomics in the various fields of Pediatric Endocrinology, including Inborn Errors of Metabolism, type 1 diabetes, Adrenal Disease, Metabolic Syndrome and Thyroid disease, ranging from neonatal screening, accurate diagnosis, early recognition of specific at-risk populations for the prevention of disease manifestation or future complications.

Irritable bowel syndrome (IBS) is a chronic gastrointestinal disorder with high incidence, and great heterogeneity of symptoms. Numerous factors are correlated with IBS development; however, the pathophysiology is not yet clear. In addition, there is no appropriate diagnostic tool available. The aim of this study was the identification of protein expression alterations in IBS patients compared to healthy individuals. Serum samples from 30 IBS patients (10 with IBS-Diarrhea, 10 IBS-Constipation and 10 IBS-Mixed) and 10 healthy individuals were subjected to proteomic analysis by 2-dimensional gel electrophoresis. Following evaluation of densitometrical data, protein spots exhibiting differential expression among the groups, were further characterized by matrix-assisted laser desorption tandem time-of-flight mass spectrometer and the results were confirmed by Western blot analysis. Eight significantly different expressed proteins were identified. Seven of them were overexpressed in IBS cases and only one was overexpressed in healthy individuals. These proteins were also differently expressed between the three IBS subgroups. IBS-D group overexpressed immunoglobulin light chain Lambda (LAC3) and apolipoprotein E (APOE), IBS-C group overexpressed apolipoprotein H (APOH) and collagen alpha-1 (XIV) chain (COEA1), and IBS-M group and healthy individuals overexpressed retinol-binding protein 4 (RET4). Our results show a different serum protein profile of IBS patients compared to healthy controls. Understanding the role of these eight proteins which are differently expressed in IBS patients, may contribute to a better clarification of IBS pathogenesis and to patient's stratification. Significance Irritable bowel syndrome (IBS) is a chronic gastrointestinal disorder with high incidence and great heterogeneity of symptoms without any appropriate diagnostic tool available. Eight significantly different expressed proteins were identified. Seven of them were overexpressed in IBS cases and only one was expressed in healthy individuals. These proteins were also differently expressed between the three IBS subgroups. Our results show that there is a different serum proteome signature in IBS compared to healthy individuals, as well as in IBS subgroups that could be used in the future for patient's stratification and as a diagnostic tool.

Mass spectrometry (MS) has dominated over other protein analysis methods that aspire to deliver rapid and sensitive protein annotation, due to its ability to acquire high-content biological information from samples of great complexity. Routinely, in-depth analysis of complex biological samples, such as total cell lysates, relies on the high separation power of two-dimensional liquid chromatography-tandem MS (2D LC–MS/MS), often combined with protein pre-fractionation. However, on the basis of recent advances in chromatographic and MS instrumentation, one-dimensional (1D) LC–MS/MS approaches have become the method-of-choice for high-volume/high-throughput protein experiments. Thousands of proteins can be identified in single-run LC–MS/MS experiments. In the present study a 1D LC–MS/MS approach was applied on whole-cell lysates of WM-266-4 human cells leading to identification of more than 5,300 protein groups, 6,000 proteins and 22,00 peptides, in a single run. Using no pre-fractionation steps, method optimization was achieved through experimentation on lysis and protein extraction solutions, as well as nLC gradient parameters.

Milk and dairy products are a major functional food group of growing scientific and commercial interest due to their nutritional value and bioactive &ldquo, load&rdquo, A major fraction of the latter is attributed to milk&rsquo, s rich protein content and its biofunctional peptides that occur naturally during digestion. On the basis of the identified proteome datasets of milk whey from sheep and goat breeds in Greece and feta cheese obtained during previous work, we applied an in silico workflow to predict and characterise the antimicrobial peptide content of these proteomes. We utilised existing tools for predicting peptide sequences with antimicrobial traits complemented by in silico protein cleavage modelling to identify frequently occurring antimicrobial peptides (AMPs) in the gastrointestinal (GI) tract in humans. The peptides of interest were finally assessed for their stability with respect to their susceptibility to cleavage by endogenous proteases expressed along the intestinal part of the GI tract and ranked with respect to both their antimicrobial and stability scores.

Over the past 30 years there has been a growing interest to unravel the dynamic framework of the milk proteome, and now that available technology is mature enough to enable techniques of protein fractionation and identification, this process is on-going. Due to its rarity and unique biological traits, as well as its growing financial value, milk of dairy Greek animals is continuously attracting interest from both the scientific community and industry. In the present study we employed cutting-edge proteomics methodologies to investigate and characterize, in depth, the proteome of whey from all pure-breed Greek sheep and goats. A mean of > 500 protein groups were identified in whey from each breed of each animal species, reporting for the first time the proteome dataset of this precious biological material. Given its high nutritional value, the protein properties exposed herein will govern future steps in optimizing characteristics and features of sheep and goat milk products. Significance In the present study we employed cutting-edge proteomics methodologies to investigate and characterize, in depth, the proteome of milk from all pure-breed Greek sheep and goats. A mean of > 500 protein groups were identified in milk whey from each breed of each animal species, reporting for the first time the proteome dataset of this precious biological material. Given its high nutritional value, the protein properties exposed herein will govern future steps in optimizing characteristics and features of sheep and goat milk products.

Pediatric brain tumors (PBTs) are the most common solid malignancies in childhood and continue to pose a serious burden to modern societies. Existing treatments impose debilitating effects on the developing child, highlighting the need for molecularly targeted treatments with reduced toxicity, as well as the necessity of markers that reliably assess efficacy of, and tumor response to targeted-therapies of PBTs. On this regard advances in technologies of protein identification and quantification, the large-scale, high-throughput investigation of the proteome, as well the newly-emerging field of "proteogenomics" aim to further our knowledge towards understanding the molecular pathophysiology of PBTs. This mini review article presents all updates on knowledge produced and published during the last years on PBT research derived from "omics" technologies, mainly involving protein research and proteomics.

To explore the photo-therapeutic capacity of UV radiation in solid tumors, we herein employed an nLC-MS/MS technology to profile the proteomic landscape of irradiated WM-266-4 human metastatic-melanoma cells. Obtained data resulted in proteomic catalogues of 5982 and 7280 proteins for UVB- and UVC-radiation conditions, respectively, and indicated the ability of UVB/C-radiation forms to eliminate metastatic-melanoma cells through induction of synergistically operating programs of apoptosis and necroptosis. However, it seems that one or more WM-266-4 cell sub-populations may escape from UV-radiation's photo-damaging activity, acquiring, besides apoptosis tolerance, an EMT phenotype that likely offers them the advantage of developing resistance to certain chemotherapeutic drugs. Low levels of autophagy may also critically contribute to the selective survival and growth of UV-irradiated melanoma-cell escapers. These are the cells that must be systemically targeted with novel therapeutic schemes, like the one of UV radiation and Irinotecan herein suggested to be holding strong promise for the effective treatment of metastatic-melanoma patients. Given the dual nature of UV radiation to serve as both anti-tumorigenic and tumorigenic agent, all individuals being subjected to risk factors for melanoma development have to be appropriately informed and educated, in order to integrate the innovative PPPM concept in their healthcare-sector management. Significance This study reports the application of nLC-MS/MS technology to deeply map the proteomic landscape of UV-irradiated human metastatic-melanoma cells. Data bioinformatics processing led to molecular-network reconstructions that unearthed the dual nature of UV radiation to serve as both anti-tumorigenic and tumorigenic factor in metastatic-melanoma cellular environments. Our UV radiation-derived “photo-proteomic” atlas may prove valuable for the identification of new biomarkers and development of novel therapies for the disease. Given that UV radiation represents a major risk factor causing melanoma, a PPPM-based life style and clinical practice must be embraced by all individuals being prone to disease's appearance and expansion.

Background/aim Medulloblastoma (MBL), an archetypal primitive neuroectodermal tumor of the cerebellum, is the most common pediatric central nervous system malignancy representing approximately 20% of all childhood brain tumors. Herein, we report on a new xenotransplantable tumor cell line, derived from a 6-year-old female patient with cerebellar medulloblastoma, and the completele proteome molecular characterization of subsequent tumors from MBL xenotrasplanted mice. Materials and methods Tumors were grown in nude mice as subcutaneous xenografts (MBLX) composed of small round cells with hyperchromatic nuclei and scant cytoplasm. Tumor specimen were extracted from animals upon their sacrifice and their molecular proteomic content was analyzed by 2-DE coupled to MALDI-TOF MS analysis. Results Altogether 350 single-gene products were identified through the current approach, reported as the MBLX database. Conclusion This new xenotransplantable tumor model, offers the scientific community valuable insight on the validity of xenografts altogether, while providing the means for a novel experimental model towards the study of human MBL.

To identify potential biomarkers in the 1st trimester of pregnancy for the identification of women destined to develop early onset preeclampsia (EOPE).Blood samples were obtained from pregnant women at 11-13 weeks of gestation. Women were followed up until delivery. Five samples from EOPE complicated pregnancies and 5 from unaffected ones were analysed using 2-DE and MALDI-TOF-TOF MS/MS. The altered expression of selected proteins was verified by ELISA in an extended sample cohort.Twelve proteins were differentially expressed in the plasma of women who subsequently developed EOPE as compared to controls. Alpha-1-antitrypsin (A1AT), CD5 antigen-like molecule (CD5L) Keratin, type I cytoskeletal 9 (K1C9), Myeloid cell nuclear differentiation antigen (MNDA), Transferrin (TRFE) and Vitamin D-binding protein (VTDB) were up-regulated with fold changes 3.14, 2.18, 1.53, 1.53, 4.26 3.38 respectively, whereas Alpha-2-HS-glycoprotein (FETUA), Beta-2-glycoprotein 1 (APOH), Complement factor B (CFAB), Haptoglobin (HPT), Vitronectin (VTNC) and Zinc-alpha-2-glycoprotein (ZA2G) were down-regulated with fold changes -0.38, -0.76, -0.24, -0.47, -0.23, and -0.50 respectively. The down-regulation of APOH, VTNC and HPT was verified using ELISA.The differentially expressed proteins represent potential biomarkers for the early screening for EOPE. Follow-up experiments however are necessary for evaluation.

The Spartathlon race (brisk walking a distance of 246 km in less than 36 h) was employed as a model of severe physical stress to investigate proteomic alterations in the plasma of athletes at the start (Athens) and finish (Sparta) of the race, as well as 48 h after the race (Post). The athletes' plasma was analyzed by 2D gel electrophoresis (2-DE) and the differentially expressed proteins were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). The ProteoSeek™ Albumin/IgG removal kit and the ProteoMiner™ enrichment kit were utilized to detect medium- and low-abundance proteins, whose expression may be masked due to high-abundance proteins. Our results were confirmed by Western blot and biochemical analyses. Overall fifty-two proteins were differentially expressed between the starting point, the finishing line and two days after the end of the race. Of these, thirty proteins were involved in inflammation, while the rest concerned anti-oxidation, anti-coagulation and iron and vitamin D transport. These results indicate that prolonged physical stress affects circulating stress-related proteins, which might be employed as biomarkers of stress-related diseases. Biological significance The current study employed the Spartathlon, as a model of prolonged endurance exercise, to identify and isolate putative biomarkers of inflammation under extreme physical stress conditions. These protein quantitative variations may pave the way to exploration and understanding of stress-related physiological processes, the stress response itself and diseases whose onset appears to be linked to stress.

Objective was the differential quantitative proteomics study of ovine mastitis induced by Mannheimia haemolytica; clinical, microbiological, cytological and histopathological methods were employed for confirmation and monitoring. Proteins were separated by two-dimensional gel electrophoresis (2-DE) for all samples and differentially abundant proteins were identified by mass spectrometry; comparisons were performed with pre- (blood, milk) and post- (milk of contralateral gland) inoculation findings. Animals developed mastitis, confirmed by isolation of challenge strain and increase of neutrophils in milk and by histopathological evidence. In blood plasma, 33 differentially abundant proteins (compared to findings before challenge) were identified: 6 with decrease, 13 with new appearance and 14 with varying abundance. In a post-challenge milk whey protein reference map, 65 proteins were identified; actin cytoplasmic-1, beta-lactoglobulin-1/B, cathelicidin-1 predominated. Further, 89 differentially abundant proteins (compared to findings before challenge) were identified: 18 with decrease, 53 with new appearance, 3 with increase and 15 with varying abundance; 15 proteins showed status changes in blood plasma and milk whey. Differential abundance from inoculated and contralateral glands revealed 74 proteins only from the inoculated gland. Most differentially abundant proteins in milk whey were involved in cell organisation and biogenesis (n = 17) or in inflammatory and defence response (n = 13). Significance The proteomes of blood and milk from ewes with experimental mastitis caused by Mannheimia haemolytica and the differential proteomics in sequential samples after challenge are presented for the first time. This is the first detailed proteomics study in M. haemolytica-associated mastitis in ewes. An experimental model fully simulating natural mastitis has been used. Use of experimentally induced mastitis minimised potential variations and allowed consistency of results. The study included evaluation of changes in blood plasma and milk whey. Protein patterns have been studied, indicating with great accuracy changes that had occurred as part of the disease process and development, during the acute phase of infection. Relevant protein-protein interactions were studied. The entirety of proteomics findings has suggested that affected ewes had mounted a defence response that had been regulated by many proteins (e.g., cathelicidins, haptoglobin, serum amyloid A) and through various pathways (e.g., acute phase response, binding and transporting significant ions and molecules); these were interdependent at various points. Potential biomarkers have been indicated for use in diagnostic assays of mastitis.

Background/aim Proteomics based on high-resolution mass spectrometry (MS) is the tool of choice for the analysis of protein presence, modifications and interactions, with increasing emphasis on the examination of tumor tissues. Application of MS-based proteomics offers a detailed picture of tumor tissue characteristics, facilitating the appreciation of different tumor entities, whilst providing reliable and fast results for therapeutic marker targeting and prognostic factor assessment. Through use of the high analytical resolution of nano-high-pressure liquid chromatography (nanoHPLC) and the high resolution of an Orbitrap Elite mass spectrometer, the present study aimed to provide knowledge on the proteome of the generally unknown entity of pediatric ependymal tumors. Materials and methods Ten resected specimens of childhood ependymoma were analyzed through a one-dimensional (1D) nanoLC-MS/MS approach. Method optimization steps were undertaken for both the sample preparation/protein extraction procedure and LC parameters, aiming to achieve the highest possible identification rates. Results Following method optimization, each nanoLC-MS/MS run resulted in identification of more than 5,000 proteins and more than 25,000 peptides for every analyzed sample, thus detailing the greater part of the ependymoma proteome. Identified proteins were found to spread throughout all known tumor categories regarding their molecular function and subcellular localization. Conclusion Through the proposed nanoLC-MS/MS method herein we report, for the firs time, the ependymoma proteome database. A large number of similarities regarding proteome content are revealed compared to other two pediatric brain tumor entities; astrocytomas and medulloblastomas. Furthermore, through our approach, the majority of currently proposed markers for ependymoma (e.g. nucleolin, nestin, Ki67 and laminin subunit A2) as well as all major key players of the phosphoinositide 3-kinase pathway (seemingly implicated in ependymoma), were definitely detected.

Cutaneous melanoma is a malignant tumor of skin melanocytes that are pigment-producing cells located in the basal layer (stratum basale) of epidermis. Accumulation of genetic mutations within their oncogenes or tumor-suppressor genes compels melanocytes to aberrant proliferation and spread to distant organs of the body, thereby resulting in severe and/or lethal malignancy. Metastatic melanoma's heavy mutational load, molecular heterogeneity and resistance to therapy necessitate the development of novel biomarkers and drug-based protocols that target key proteins involved in perpetuation of the disease. To this direction, we have herein employed a nano liquid chromatography-tandem mass spectrometry (nLC-MS/MS) proteomics technology to profile the deep-proteome landscape of WM-266-4 human metastatic melanoma cells. Our advanced melanoma-specific catalogue proved to contain 6,681 unique proteins, which likely constitute the hitherto largest single cell-line-derived proteomic collection of the disease. Through engagement of UNIPROT, DAVID, KEGG, PANTHER, INTACT, CYTOSCAPE, dbEMT and GAD bioinformatics resources, WM-266-4 melanoma proteins were categorized according to their sub-cellular compartmentalization, function and tumorigenicity, and successfully reassembled in molecular networks and interactomes. The obtained data dictate the presence of plastically inter-converted sub-populations of non-cancer and cancer stem cells, and also indicate the oncoproteomic resemblance of melanoma to glioma and lung cancer. Intriguingly, WM-266-4 cells seem to be subjected to both epithelial-to-mesenchymal (EMT) and mesenchymal-to-epithelial (MET) programs, with 1433G and ADT3 proteins being identified in the EMT/MET molecular interface. Oncogenic addiction of WM-266-4 cells to autocrine/paracrine signaling of IL17-, DLL3-, FGF(2/13)- and OSTP-dependent sub-routines suggests their critical contribution to the metastatic melanoma chemotherapeutic refractoriness. Interestingly, the 1433G family member that is shared between the BRAF- and EMT/MET-specific interactomes likely emerges as a novel and promising druggable target for the malignancy. Derailed proliferation and metastatic capacity of WM-266-4 cells could also derive from their metabolic addiction to pathways associated with glutamate/ammonia, propanoate and sulfur homeostasis, whose successful targeting may prove beneficial for advanced melanoma-affected patients.

Childhood pilocytic astrocytoma is the most frequent brain tumor affecting children. Proteomics analysis is currently considered a powerful tool for global evaluation of protein expression and has been widely applied in the field of cancer research. In the present study, a series of proteomics, genomics, and bioinformatics approaches were employed to identify, classify and characterize the proteome content of low-grade brain tumors as it appears in early childhood. Through bioinformatics database construction, protein profiles generated from pathological tissue samples were compared against profiles of normal brain tissues. Additionally, experiments of comparative genomic hybridization arrays were employed to monitor for genetic aberrations and sustain the interpretation and evaluation of the proteomic data. The current study confirms the dominance of MAPK pathway for the childhood pilocytic astrocytoma occurrence and novel findings regarding the ERK-2 expression are reported.

The emerging field of bioinformatics in proteomics is introducing new algorithms in order to handle large and heterogeneous datasets and improve the knowledge-discovery process. Management systems, software construction and application, database population and leverage, as well as computed prediction, have crafted bioinformatics into a valuable tool for basic research. Human reproduction is one of many fields proteomics has been extensively studying over the last decade, accumulating complex experimental data at a rate far exceeding the ability to assimilate them. Transformation of the rapidly proliferating quantities of experimental information into a usable form in order to facilitate their analysis is a challenging task. On this track, bioinformatics, an essential part of proteomics research, aspires to amend inquiries into a better manipulated, a better handled and a better understood form so as to enhance existing knowledge expansion.

Turner syndrome (TS) is the most common sex chromosome abnormality in females, caused by the complete or partial absence of one X chromosome. To identify biomarkers for TS, we compared the protein composition of maternal plasma samples from pregnant women with normal and TS fetuses, using a proteomic approach consisting of 2D-E separation and MS analysis for the identification of the differentially expressed proteins. Samples were routinely obtained in the second trimester of pregnancy, stored, and used after prenatal determination of the fetal karyotype. Nine proteins (C1S, CO3, CLUS, AFAM, HABP2, IGHA1, HPT, SHBG, and CD5L) were significantly increased in the plasma of women carrying TS fetuses, whereas KNG1, IGJ, and TTHY were decreased. Identified proteins were further evaluated by immunoblot analysis while functional network association was carried out to asses significance. The identification of specific biomarkers may facilitate the development of noninvasive prenatal diagnosis and improve our understanding of the pathology of TS. Nevertheless, testing a larger cohort of pregnant women is necessary to evaluate the relevance of the reported findings.

The aim of this study was to investigate the progression of myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML) and to provide additional data regarding the proteomic analysis of AML. The protein profiles obtained were correlated to cytogenetic and molecular analyses. Bone marrow (BM) and peripheral blood (PB) samples were obtained during MDS diagnosis, at MDS transformation to AML, at de novo AML diagnosis and 3 months following treatment. As controls, non-leukemic pediatric patients were studied. Cytogenetic and molecular analyses were carried out by G banding and polymerase chain reaction followed by sequencing, respectively. Differential proteomic analysis was performed by two-dimensional gel electrophoresis and protein identification by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. No significant correlations were noted between protein patterns and cytogenetic or molecular analyses. Certain suppressor genes, metabolic enzymes, immunoglobulins and actin-binding proteins were differentially expressed by BM or PB plasma and cell lysates compared to controls. The obtained data showed that vitamin D and gelsolin played contradicting roles in contributing and restraining leukemogenesis, while MOES, EZRI and AIFM1 could be considered as biomarkers for AML.

Klinefelter syndrome is a sex chromosomal abnormality (47, XXY karyotype), occurring approximately in 1 in 1000 male live births. In the present study proteomic analysis was performed in twelve 2nd trimester amniotic fluid samples, eight coming from pregnancies with normal males and four with Klinefelter syndrome foetuses, as shown by routine prenatal cytogenetic analysis. Samples were analysed by 2-DE, coupled with MALDI-TOF-MS analysis. Three proteins (Ceruloplasmin, Alpha-1-antitrypsin and Zinc-alpha-2-glycoprotein) were found to be up-regulated in samples obtained from pregnancies with Klinefelter syndrome foetuses, whereas four proteins (Apolipoprotein A-I, Plasma retinol-binding protein, Gelsolin, and Vitamin D-binding protein) were down regulated when compared to proteins detected in samples from normal foetuses. The differential expression of Ceruloplasmin, Apolipoprotein A-I and Plasma retinol-binding protein was further confirmed by immunoblotting. Since these proteins are likely to cross the placenta barrier and be detected in maternal plasma they could be used as biomarkers for the non-invasive prenatal diagnosis of Klinefelter syndrome.

The third International Conference of the Hellenic Proteomics Society, From Proteomics Research to Clinical Practice, took place in Nafplio (Greece), from 30 March to 1 April 2009. This year the conference was dedicated to the application of proteomics in clinical practice. Many scientists from different European counties participated in the conference, which made this event unique in the field of proteomics for the southeastern region of Europe. Extensive presentations and discussions covered nearly every aspect of the modern point of view regarding the application of proteomics in various diseases, the quantitative peptidomics and proteomics approaches, and the advances of methodologies for biomarker discovery and validation.

We have identified the gene product of locus 766 in the transmembrane head region (TMH/Inc-region) in the Chlamydophila abortus genome by using mass spectrometry and a monoclonal antibody that reacted with the inclusion membrane. The identified protein at 32 kDa, termed Inc766, formed highly stable oligomers when solubilized in the absence of β-mercaptoethanol. These oligomers were resistant to SDS, to heat denaturation and to 8 M urea, but very sensitive to β-mercaptoethanol, consistent with conformations resulting from protein–protein interactions stabilized through disulphide bonds. Mass spectrometry analysis of immunoprecipitated infected cell lysates indicated that a dimer at 56 kDa was the most prominent form in solution. Cross-linking with DSP provided supporting evidence for the formation of oligomers in situ . Inc766 was expressed at 20–24 h post infection and its localization pattern in the extra-inclusion space was common in all C. abortus strains tested. Taken together, Inc766 displays unique biochemical and cellular features not encountered in other Incs from other Chlamydiaceae species. Future studies of the particular characteristics especially the interactive properties of Inc766 should contribute to our understanding of the relationship of the different chlamydial species with their respective hosts.

Proteomic analysis is widely used for the detection of diagnostic markers. In the present study amniotic fluid supernatants (AFS) from pregnancies with Down syndrome (DS) fetuses and from chromosomally normal fetuses in the 17th week of gestation were analyzed by 2-DE. Gel comparison revealed significant differences in the two groups. Spots with different expression levels were excised and proteins were identified by MALDI-MS and nano-ESI-MS/MS. Splicing factor arginine/serine-rich 4 (SFRS4; Q08170) was present only in AFS from DS fetuses and completely absent in the control group. Quantitative differences were detected for alpha-1-microglobulin (AMBP; P02760), collagen alpha 1 (I) chain (CO1A1; P02452), collagen alpha 1 (III) chain (CO3A1; P02461), collagen alpha 1 (V) chain d (CO5A1; P20908), and basement membrane-specific heparin sulfate proteoglycan core protein (PGBM; P98160). These proteins were increased in cases with DS, whereas protein IBP-1 (P08833) was decreased by 40% compared with chromosomally normal fetuses. Four proteins, CO1A1, CO3A1, CO5A1, and PGBM, appeared as fragments. As differentially expressed proteins were present in all pregnancies with DS tested, they may represent useful potential markers for prenatal diagnosis. However, for protein biomarkers to be of any clinical utility, systematic analysis of the maternal serum should be conducted.

HeLa cells are widely used for all kinds of in vitro studies in biochemistry, biology and medicine. Knowledge on protein expression is limited and no comprehensive study on the proteome of this cell type has been reported so far. We applied proteomics technologies to analyze the proteins of the HeLa cell line. The proteins were analyzed by two-dimensional (2D) gel electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry (MS) on the basis of peptide mass fingerprinting, following in-gel digestion with trypsin. Approximately 3000 spots, excised from six two-dimensional gels, were analyzed. The analysis resulted in the identification of about 1200 proteins that were the products of 297 different genes. The HeLa cell database includes proteins with important functions and unknown functions, representing today one of the largest two-dimensional databases for eukaryotic proteomes and forming the basis for future expressional studies at the protein level.

CNS tumors are the leading cause of cancer-related death in children. Medulloblastoma is the commonest pediatric CNS malignancy, wherein, despite multimodal therapy with surgery, radiation, and chemotherapy, 5 year survival rates merely approach 60%. Until present, gene expression and cytogenetic studies have produced contradicting findings regarding the molecular background of the specific disease. Through integration of genomics, bioinformatics, and proteomics, the current study aims to shed light at the proteomic-related molecular events responsible for MBL pathophysiology, as well as to provide molecular/protein/pathway answers concerning tumor-onset. Experiments were performed on tissues collected at surgery. With 17p loss being the commonest chromosomal aberrance observed in our sample set, array-CGH were employed to first distinguish for 17p-positive cases. 2-DE coupled to mass spectrometry identification exposed the MBL-specific protein profile. Protein profiles of malignant tissues were compared against profiles of normal cerebellar tissues, and quantitative protein differences were determined. Bioinformatics, functional and database analyses, characterization, and subnetwork profiling generated information on MBL protein interactions. Key molecules of the PI3K/mTOR signaling network were identified via the techniques applied herein. Among the findings IGF2, PI3K, Rictor, MAPKAP1, S6K1, 4EBP1, and ELF4A, as part of the IGF network (implicating PI3K/mTOR), were founded to be deregulated.

Pediatric tumors of the CNS are the leading cause of cancer-related mortality in children. In pediatric pathology, brain tumors constitute the most frequent solid malignancy. An unparalleled outburst of information in pediatric neuro-oncology research has been witnessed over the last few years, largely due to increased use of high-throughput technologies such as genomics, proteomics and meta-analysis tools. Input from these technologies gives scientists the advantage of early prognosis assessment, more accurate diagnosis and prospective curative intent in the pediatric brain tumor clinical setting. The present review aims to summarize current knowledge on research applying proteomics techniques or proteomics-based approaches performed on pediatric brain tumors. Proteins that can be used as potential disease markers or molecular targets, and their biological significance, are herein listed and discussed. Furthermore, future perspectives that proteomics technologies may offer regarding this devastating disorder are presented.

Laterality is a basic characteristic of the brain which is detectable early in life. Although early experiences affect laterality of the mature brain, there are no reports on their immediate neurochemical effects during neonatal life, which could provide evidence as to the mechanisms leading to the lateralized brain. In order to address this issue, we determined the differential protein expression profile of the left and right hippocampus of 13-day-old rat control (CTR) pups, as well as following exposure to an early experience involving either receipt (RER) or denial (DER) of the expected reward of maternal contact. Proteomic analysis was performed by 2-dimensional polyacrylamide gel electrophoresis (PAGE) followed by mass spectroscopy. The majority of proteins found to be differentially expressed either between the three experimental groups (DER, RER, CTR) or between the left and right hemisphere were cytoskeletal (34%), enzymes of energy metabolism (32%), and heat shock proteins (17%). In all three groups more proteins were up-regulated in the left compared to the right hippocampus. Tubulins were found to be most often up-regulated, always in the left hippocampus. The differential expression of β-tubulin, β-actin, dihydropyrimidinase like protein 1, glial fibrillary acidic protein (GFAP) and Heat Shock protein 70 revealed by the proteomic analysis was in general confirmed by Western blots. Exposure to the early experience affected brain asymmetry: In the RER pups the ratio of proteins up-regulated in the left hippocampus to those in the right was 1.8, while the respective ratio was 3.6 in the CTR and 3.4 in the DER. Our results could contribute to the elucidation of the cellular mechanisms mediating the effects of early experiences on the vulnerability for psychopathology, since proteins shown in our study to be differentially expressed (e.g. tubulins, dihydropyrimidinase like proteins, 14-3-3 protein, GFAP, ATP synthase, α-internexin) have also been identified in proteomic analyses of post-mortem brains from psychiatric patients.

We previously showed in a human T cell line (CEM-C12 cells) that Cd2+ induced gene expression of stress proteins, metallothionein-IIA and heat shock protein 70 in a time- and dose-dependent manner. In the present study, CEM-C12 cells were pretreated for 24 h with 1 microM Cd2+ and then challenged with toxic concentrations of this metal. We found that maximal expression of the metallothionein-IIA and heat shock protein 70 genes was increased and this maximal level occurred at higher Cd2+ toxic concentrations. Actinomycin D chase experiments indicated that Cd2+ pretreatment did not modify metallothionein-IIA mRNA stability. The modulatory effect of Cd2+ pretreatment was dose-dependent from 100 pM to 1 microM. Such pretreatment also enhanced resistance to Cd2+ toxicity. Finally, verapamil, a calcium/potassium channel blocker displaced the dose-response curve for Cd2+ toxicity as well as metallothionein-IIA and heat shock protein 70 gene expression to higher Cd2+ concentrations.

Biological systems demonstrate asymmetry, while lateralization has been observed from humans to lower animals structurally, functionally and behaviorally. This may be derived from evolutionary, genetic, developmental, epigenetic and pathologic factors. However, brain structure and function is complex, and macroscopic or microscopic asymmetries are hard to discern from random fluctuations. In this article, we discuss brain laterality and lateralization, beginning with a brief review of the literature on brain structural and functional asymmetries. We conclude with methods to detect and quantify asymmetry, focusing on neuroproteomics, for retrieval of protein-expression patterns, as a method of diagnosis and treatment monitoring. We suggest inter-hemispheric differential proteomics as a valid method to assess the experimental and biological variations in the healthy brain, and neurologic and neuropsychiatric disorders.

The evidence on the mechanisms underlying schizophrenia seems to support a series of divergent possibilities that may play a contributory role in the disease pathogenesis. Hypotheses regarding disease mechanisms range from structural and neurodevelopmental abnormalities to neurotransmitter deficits and genetic predisposition. Nevertheless, little progress has been made in the field of schizophrenia with respect to the key pathophysiological processes, and this is a fundamental barrier to identifying or predicting the therapeutic value of novel drug targets. All multi-omics approaches, including proteomics, would be perfectly suited to such a complex disease as a means of tackling the widening chasm between the aforementioned hypotheses. Proteomic studies have been performed in a variety of tissues and biological fluids, from post-mortem CNS tissue to cerebrospinal fluid and blood, and in recently generated mutant animal models with schizophrenia-like phenotypes. In this review, we present an overview of several proteomic studies in the field of schizophrenia research and discuss how proteomics could assist with identifying schizophrenia markers more efficiently, covering both clinical and basic research.

Proteomic technologies represent new strategies towards high-throughput, simultaneous analysis of thousands of biological molecules leading to the discovery of biomarkers for early diagnosis, prognosis and prediction of pregnancy outcome. Proteomics have additional relevance in understanding pathophysiology and the development of molecularly targeted therapeutics. Comparison of normal human amniotic fluid proteome with that coming from pregnancies carrying fetuses with chromosomal abnormalities facilitated the detection of panels of potential biomarkers for prenatal detection of fetal aneuploidies. Candidate biomarkers for the early prediction of preeclampsis are also available, while four biomarkers (defensins-2 and -1, calgranulin-C, and calgranulin-A), which were called the «MR score», can quickly and accurately detect potentially dangerous infections and predict premature birth. Researchers remain hopeful that proteomic studies will allow for the identification of either one protein marker or of a panel of markers for prenatal detection of fetal aneuploidies and pregnancy complications that could be usefully employed for diagnostic purposes or improvement of the current screening methods. For maximum predictive power however, biomarkers should be selected for further comparative analysis of expression and structural modifications in large numbers of samples from chromosomally normal and abnormal pregnancies obtained from different populations.

Proteomic analysis of amniotic fluid cells may lead to the discovery of novel markers for embryonic abnormalities. A two-dimensional database for proteins of normal human amniotic fluid cells was constructed. The amniotic fluid cell extract was analyzed by two-dimensional gel electrophoresis and the proteins were identified by matrix-assisted laser desorption ionisation-time of flight-mass spectrometry. The database comprises 432 different gene products, which are in the majority enzymes, structural proteins, heat shock proteins, and proteins related to signal transduction. The obtained data show that the amniotic fluid population maybe either heterogeneous, originating from different fetal compartments and embryo tissues or is still pluripotent. Many proteins which are known to belong to certain cell types were found in the amnion cell fluid. This indicates that some types of fetal cells are already differentiated at the time of amniocentesis (about the 16(th) week of gestation). Moreover, the finding of proteins highly expressed in embryonic stem cells suggests that amniotic fluid could be used as a cell pool for transplantation therapy.

We constructed a reference two-dimensional protein map for horse (Equus caballus) serum. The serum proteins were separated by two-dimensional electrophoresis (2-DE); 29 different gene products were identified. Proteins represented by 25 spots/spot groups were identified by tandem nanoelectrospray mass spectrometry (MS), four by matrix-assisted laser desorption ionization time-of-flight (TOF) MS and one was sequenced by TOF-TOF technology. The identities of four proteins were deduced by similarity to the human plasma protein database. In selected cases, i.e. the immunoglobulins, immunoblotting with specific antibodies provided additional information about the respective proteins. Albumin was detected as the full-length protein and as fragments of various sizes. Spots representing products of different mass and charge were also detected for alpha1-antitrypsin, haptoglobin and transthyretin. Thus, despite the fact that the Equus caballus genome is incompletely characterized, we were able to identify almost all moderate to high abundance proteins stained in the serum 2-DE pattern.

The hippocampus is crucial in memory storage and retrieval and plays an important role in stress response. In humans, the CA1 area of hippocampus is one of the first brain areas to display pathology in Alzheimer's disease. A comprehensive analysis of the hippocampus proteome has not been accomplished yet. We applied proteomics technologies to construct a two-dimensional database for rat brain hippocampus proteins. Hippocampus samples from eight months old animals were analyzed by two-dimensional electrophoresis and the proteins were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The database comprises 148 different gene products, which are in the majority enzymes, structural proteins and heat shock proteins. It also includes 39 neuron specific gene products. The database may be useful in animal model studies of neurological disorders.

Proteomics offers unique possibilities to investigate changes in the levels and modifications of proteins involved in the pathomechanisms of diseases and toxic events. However, search for potential drug targets and disease or toxicity markers is limited by the fact that mainly the high-abundance, hydrophilic proteins are visualized in two-dimensional gels. Here we studied the enrichment of rat liver cytosolic proteins by preparative electrophoresis. Preparative electrophoresis was performed with the PrepCell apparatus in the presence of 0.1% lithium dodecyl sulfate. Lithium dodecyl sulfate was exchanged against agents compatible with isoelectric focusing prior to the two-dimensional gel electrophoresis. Proteins were identified from two-dimensional gels by matrix-assisted laser desorption ionization time-of-flight mass specrometry. Low- and middle-size proteins and low-abundance proteins, which had not been found before, were enriched by preparative electrophoresis. The present study represents a contribution of proteomics in the quantification of differences in the levels of low-abundance liver proteins in toxicity studies.