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none 9 no Ovis aries papillomavirus 3 (OaPV3) is an epidermotropic PV reported in sheep cutaneous squamous cell carcinoma (SCC). The presence of OaPV3 DNA and its transcriptional activity in cutaneous SCC, as well as its in vitro transforming properties, suggest a viral etiology for this neoplasm. Nevertheless, the reactome associated with viral-host interaction is still unexplored. Here, we investigated and compared the proteomic profiles of OaPV3-positive SCCs, OaPV3-negative SCCs, and non-SCC samples by liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis, bioinformatics tools, and immunohistochemistry (IHC). OaPV3-positive SCCs (n = 3), OaPV3-negative SCCs (n = 3), and non-SCCs samples (n = 3) were subjected to a shotgun proteomic analysis workflow to assess protein abundance differences among the three sample classes. Proteins involved in epithelial cell differentiation, extracellular matrix organization, and apoptotic signaling showed different abundances in OaPV3-positive SCCs tissues (P ≤ 0.05) when compared to the other tissues. Cytokeratin 13 (CK 13) was among the most increased proteins in OaPV3-positive SCC and was validated by immunohistochemistry on 10 samples per class, confirming its potential as a biomarker of OaPV3 infection in SCC. Collectively, results provide a preliminary insight into the reactome associated with viral-host interaction and pave the way to the development of specific biomarkers for viral-induced sheep SCC. none Vitiello V.; Burrai G.P.; Pisanu S.; Cacciotto C.; Addis M.F.; Alberti A.; Antuofermo E.; Cubeddu T.; Pirino S. Vitiello V.; Burrai G.P.; Pisanu S.; Cacciotto C.; Addis M.F.; Alberti A.; Antuofermo E.; Cubeddu T.; Pirino S.

The aim of this work was to valorize the by-product derived from the ricotta cheese process (scotta). In this study, ovine scotta was concentrated by ultrafiltration and then subjected to enzymatic hydrolyses using proteases of both vegetable (4% E:S, 4 h, 50 °C) and animal origin (4% E:S, 4 h, 40 °C). The DPP-IV inhibitory, antioxidant, and antibacterial activities of hydrolysates from bromelain (BSPH) and pancreatin (PSPH) were measured in vitro. Both the obtained hydrolysates showed a significantly higher DPP-IV inhibitory activity compared to the control. In particular, BSPH proved to be more effective than PSPH (IC50 8.5 ± 0.2 vs. 13 ± 1 mg mL−1). Moreover, BSPH showed the best antioxidant power, while PSPH was more able to produce low-MW peptides. BSPH and PSPH hydrolysates showed a variable but slightly inhibitory effect depending on the species or strain of bacteria tested. BSPH and PSPH samples were separated by gel permeation chromatography (GPC). LC-MS/MS analysis of selected GPC fractions allowed identification of differential peptides. Among the peptides 388 were more abundant in BSPH than in the CTRL groups, 667 were more abundant in the PSPH group compared to CTRL, and 97 and 75 of them contained sequences with a reported biological activity, respectively.

The milk somatic cell count (SCC) is a standard parameter for monitoring intramammary infections (IMI) in dairy ruminants. In goats, however, the physiological increase in SCC occurring in late lactation heavily compromises its reliability. To identify and understand milk protein changes specifically related to IMI, we carried out a shotgun proteomics study comparing high SCC late lactation milk from goats with subclinical Staphylococcus aureus IMI and from healthy goats to low SCC mid-lactation milk from healthy goats. As a result, we detected 52 and 19 differential proteins (DPs) in S. aureus-infected and uninfected late lactation milk, respectively. Unexpectedly, one of the proteins higher in uninfected milk was serum amyloid A. On the other hand, 38 DPs were increased only in S. aureus-infected milk and included haptoglobin and numerous cytoskeletal proteins. Based on STRING analysis, the DPs unique to S. aureus infected milk were mainly involved in defense response, cytoskeleton organization, cell-to-cell, and cell-to-matrix interactions. Being tightly and specifically related to infectious/inflammatory processes, these proteins may hold promise as more reliable markers of IMI than SCC in late lactation goats. SIGNIFICANCE: The biological relevance of our results lies in the increased understanding of the changes specifically related to bacterial infection of the goat udder in late lactation. The DPs present only in S. aureus infected milk may find application as markers for improving the specificity of subclinical mastitis monitoring and detection in dairy goats in late lactation, when other widespread tools such as the SCC lose diagnostic value.

Cystic echinococcosis (CE) diagnosis by means of serological assays is hampered by the presence of parasites closely related to Echinococcus granulosus sensu lato (s.l.), responsible of the zoonotic disease and with which share cross-reacting antigens. Thus, improvements on the characterization of Echinococcus specific antigens expressed in the larval stage are required, in order to provide useful information for the development of immunological assays for the serodiagnosis of CE in sheep. Here, the proteome of the hydatid cyst fluids (HFs) of Echinococcus granulosus (hydatid fluid, EgHF) and other ovine parasites cyst fluids (CFs), Taenia hydatigena (ThCF) and Taenia multiceps (TmCF) were analyzed by a shotgun proteomic approach. Parasite and host protein profiles in the three types of cyst fluids were characterized and compared. Among the identified proteins, differential parasitic markers with serodiagnostic potential, due to their well-known immunoreactivity in human, included Ag5, AgB proteins, 8-kDa glycoproteins, hydatid disease diagnostic antigen P29 and major egg antigen P40. In particular, seven proteoforms of AgB and 8-kDa glycoprotein resulted to be the most promising diagnostic biomarkers, as they might predict CE in ovine and discriminate between different types of parasites.

The available cystic echinococcosis immunodiagnostic tools cannot assess cyst viability. Here, synthetic antigen B (AgB) subunits were structurally and immunologically characterized. They all showed propensity to self-oligomerize, similarly to native AgB, and AgB1 held promise as a marker of viability., Background Several tools have been proposed for serodiagnosis of cystic echinococcosis (CE), but none seems promising for cyst viability assessment. Antigens with stage-specific diagnostic value have been described, but few studies with well-characterized antigens and human serum samples have been performed. Antigen B (AgB) proteoforms hold promise as markers of viability, due to their differential stage-related expression and immunoreactivity. Methods Four AgB subunits (AgB1, AgB2, AgB3, AgB4) were synthesized and structurally characterized. Based on the preliminary evaluation of the subunits by western immunoblotting and enzyme-linked immunosorbent assay (ELISA), AgB1 and AgB2 were further tested in two ELISA setups and extensively validated on 422 human serum samples. Results All subunits showed a high degree of spontaneous oligomerization. Interacting residues within oligomers were identified, showing that both the N-terminal and C-terminal of each subunit are involved in homo-oligomer contact interfaces. No hetero-oligomer was identified. AgB1 and AgB2 ELISAs revealed different sensitivities relative to cyst stage. Of note, besides high specificity (97.2%), AgB1 revealed a higher sensitivity for active-transitional cysts (100% for CE1, 77.8% for CE2, 81.5% for CE3a, and 86.3% for CE3b) than for inactive cysts (41.7% for CE4 and 11.1% for CE5) and postsurgical patients (44%). Interestingly, 19 of 20 patients with spontaneously inactive cysts and 6 of 9 treated with albendazole >5 years earlier were negative on the AgB1 assay. Conclusions The structural characterization of subunits provides insights into the synthetic antigen conformation. The stage-related sensitivity of synthetic AgB1 holds promise as part of a multiantigen setting and deserves further longitudinal evaluation as marker of cyst viability.

We present a proteomic dataset generated from a human serum sample and the enriched/depleted fractions obtained by seven commercial products. This report is related to the research article entitled “Comparative evaluation of seven commercial products for human serum enrichment/depletion by shotgun proteomics” [1]. All samples were analyzed by LC-MS/MS, label free quantitation using the spectral counting approach, and Gene Ontology (GO) annotation. Protein relative abundances and functions were reported.

In veterinary medicine, assay performance is often affected by the lack of species-specific diagnostic tools. Reliable biomarkers might be identified by investigating biological fluids of the species of interest, but protein sequence databases are often incomplete and human-specific devices for reducing sample complexity might fail when applied to animal plasma. Here, seven commercial methods based on different capturing agents (anti-human antibodies, affinity ligands, mixture of antibodies and ligands, and combinatorial peptide ligand libraries) are applied to cat plasma and evaluated in terms of yield, identified proteins/ peptides, and relative abundance by high-resolution shotgun proteomics and label-free quantitation. As a result, anti-human antibody-based methods are unsatisfactory. Most fail in reducing albumin and immunoglobulins, and some lead to a substantial removal of other highly abundant proteins, probably because of nonspecific interactions. A protein A/dye ligand-based method is efficient in reducing immunoglobulins, fibrinogen, and apolipoprotein A1 and A2, but not albumin, and protein identifications do not increase. Only peptide ligand libraries flatten the dynamic range, and increased protein identification (59.0%). Albumin and immunoglobulins are successfully depleted (60.7% and 35.9%, respectively). Although further studies will be required for reinforcing our observations, this work can provide a useful guide for cat plasma pretreatment in biomarker discovery studies.

Cheese produced using raw ovine milk (R) or pasteurised ovine milk (P) was subjected to peptide extraction, characterisation by mass spectrometry and bioinformatic analysis with the aim of assessing the impact of milk pasteurisation on the final peptide profile. In total, 187 peptides arising from β-casein, αS1-casein, and αS2-casein were identified. Upon label-free quantitation, 58 peptides were found to be significantly different in the two preparations; 38 were more abundant in R and 20 were more abundant in P. Of these, 27 were unique to R and 10 were unique to P. Bioinformatic analysis by EnzymePredictor provided insights into the influence of milk pasteurisation on susceptibility of cheese proteins to proteolytic enzymes during ripening. Finally, BIOPEP analysis predicted a biological activity for 37 of the 187 identified sequences (20%), with a significantly higher abundance of peptides with immunomodulating and ACE-inhibitor properties in R cheeses.

Cathelicidins are well-characterized antimicrobial peptides (AMPs) that are present in significant amounts in mastitic milk. Neutrophils are believed to be the main producers of these AMPs, while the role of mammary epithelial cells (MECs) in their production and release is still unclear. In this work, cathelicidin production patterns were investigated in mammary tissues of ewes infected by Staphylococcus aureus, Streptococcus uberis, or Mycoplasma agalactiae, with a combined approach including immunohistochemistry, immune-colocalization, and fluorescent in situ hybridization. Our results confirm that MECs produce and release cathelicidins in response to different mastitis pathogens. As opposed to neutrophils, however, MECs do not seem to store the preformed protein precursor in their cytoplasm, but appear to synthesize and release it only upon exposure to the microorganisms. Cathelicidin production by MECs appears to occur before leukocyte influx in the milk, suggesting a role for these cells in the initial response of the mammary epithelium to microbial infection. Once in the milk, infiltrating neutrophils release massive amounts of cathelicidin by degranulation and production of neutrophil extracellular traps, acting as the main contributor for cathelicidin abundance in mastitic milk. Taken together, our results support the active contribution of MECs to cathelicidin production and release, and reinforce the value of cathelicidins as sensitive and pathogen-independent mastitis markers.

Investigating the innate immune response mediators released in milk has manifold implications, spanning from elucidation of the role played by mammary epithelial cells (MECs) in fighting microbial infections to the discovery of novel diagnostic markers for monitoring udder health in dairy animals. Here, we investigated the mammary gland response following a two-step experimental infection of lactating sheep with the mastitis-associated bacterium Streptococcus uberis . The establishment of infection was confirmed both clinically and by molecular methods, including PCR and fluorescent in situ hybridization of mammary tissues. Proteomic investigation of the milk fat globule (MFG), a complex vesicle released by lactating MECs, enabled detection of enrichment of several proteins involved in inflammation, chemotaxis of immune cells, and antimicrobial defense, including cathelicidins and calprotectin (S100A8/S100A9), in infected animals, suggesting the consistent involvement of MECs in the innate immune response to pathogens. The ability of MECs to produce and release antimicrobial and immune defense proteins was then demonstrated by immunohistochemistry and confocal immunomicroscopy of cathelicidin and the calprotectin subunit S100A9 on mammary tissues. The time course of their release in milk was also assessed by Western immunoblotting along the course of the experimental infection, revealing the rapid increase of these proteins in the MFG fraction in response to the presence of bacteria. Our results support an active role of MECs in the innate immune response of the mammary gland and provide new potential for the development of novel and more sensitive tools for monitoring mastitis in dairy animals.

The small size of goat milk fat globules (MFGs) is one of the factors contributing to the higher digestibility of goat milk compared to other milks. In this study, size, protein composition and lipid distribution of MFGs were evaluated comparatively in a popular dairy breed, Saanen, and in a minor breed, Sarda. MFGs were found to be significantly smaller in Sarda compared to Saanen goats, with average diameters of 2.73 ± 0.15 μm and 3.63 ± 0.27 μm, respectively. Raman spectroscopy revealed differences in the lipid profiles of differently sized MFGs within each breed, with MFGs of the same size class having comparable profiles between breeds. Proteomic characterization by SDS-PAGE followed by tandem mass spectrometry (GeLC–MS/MS) and label-free differential quantification highlighted significant differences in expression levels of MFG proteins from the two breeds, with a higher abundance of cytoplasmic proteins in Sarda MFGs and of membrane proteins in Saanen MFGs. Moreover, differences in the relative abundance of several major MFG proteins were observed for the two breeds. In conclusion, this study demonstrates the existence of breed-dependent differences in the lipid and protein makeup of goat MFGs, likely related to their different size distribution. This highlights once again the importance of investigating biodiversity in autochthonous and neglected breeds, which often possess valuable attributes that might be lost as a consequence of the widespread diffusion of highly productive, but more homogeneous, dairy breeds.

Johne's disease (JD) is a chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). To identify the processes activated in the sheep intestine during natural MAP infection, and to provide a panel of differential host and pathogen proteins with diagnostic and prognostic potential, a differential shotgun proteomics workflow, including mass spectrometry, label-free quantisation and pathway analysis, was applied to ileal tissues of ewes with and without JD. Out of 2889 total proteins identified, 384 were differentially expressed and 341 were expressed at a higher level in JD. On the basis of Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis, these proteins were involved in numerous relevant biological networks and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including inhibition of phagosome acidification (such as V-ATPase), bacterial invasion, leucocyte recruitment and activation, and antimicrobial activity (such as haptoglobin, lactoferrin, cathelicidins, calgranulins and interleukins). A total of 28 MAP proteins were identified, including bacterioferritin, β-lactamase and heparin-binding haemagglutinin (HBHA), a mycobacterial adhesin crucial for dissemination of infection.

Diabetes is a chronic metabolic disease which can lead to serious health problems particularly in and to the development of cardiovascular and renal complications. The aim of this study is to possibly identify distinctive molecular features in urine samples which might correlate to the progression and complications of type 1 diabetes. Diabetic patients with normo- and micro-albuminuria have been analyzed and compared to a group of control subjects. Urine proteins of control and type 1 diabetes subjects were investigated in their proteome profiles, using high-resolution two-dimensional gel electrophoresis separation and protein identifications by MALDI-TOF-MS and LC-MS/MS analysis. Proteomics analysis highlighted differential expression of several proteins between control and type 1 diabetes subjects. In particular, five proteins were found to be down-regulated and four proteins up-regulated. Lower protein representations in diabetic subjects were associated with Tamm-Horsfall urinary glycoprotein, apolipoprotein A-I, apolipoprotein E, α2-thiol proteinase inhibitor, and human complement regulatory protein CD59, while higher protein representations were found for α-1-microglobulin, zinc-α2 glycoprotein, α-1B glycoprotein, and retinol-binding protein 4. These differences were maintained comparing control subjects with type 1 diabetes normo-albuminuric and micro-albuminuric subjects. Furthermore, these proteins are correlated to glycosylated hemoglobin and microalbuminuria, confirming their role in diabetic pathology. This study gives new insights on potential molecular mechanisms associated with the complications of type 1 diabetic disease providing evidences of urine proteins potentially exploitable as putative prognostic biomarkers.

Milk fat globules (MFGs) are fat droplets released in milk as vesicles with a tripartite membrane structure. MGF proteins (MFGPs) are involved in many metabolic processes, and are recently gaining growing attention. Sheep MFGPs were extracted and characterized by two-dimensional polyacrylamide gel electrophoresis/mass spectrometry (2-DE/MS), and a reference map was established. Specifically, 29 MFGPs were successfully identified by tandem MS from 61 spots, localized in the reference map, and characterized for gene ontology. For comparison, skim milk and whey were also subjected to 2-DE/MS, achieving identification of all the major sheep milk proteins and their localization in reference maps. Reproducibility of MFGP maps was then assessed; no significant variations were detectable when comparing samples from different healthy animals at the same stage of lactation. These results provide the first 2-DE reference map of sheep MFGPs, opening the way to studies on their expression in different physiological and pathological conditions.

Bouin's solution has been used for over a century as a common fixative in several pathology laboratories worldwide. Therefore, a considerable number of Bouin-fixed paraffin-embedded (BFPE) tumor samples of various origin are available in hospital repositories as a powerful information mine for clinical investigations. To date, however, such archived tissues have not been subjected to a systematic study aimed to evaluate their potential use in proteomics. In this report, we investigated whether archival BFPE tissue specimens could be exploited for proteomic studies, upon application of protein extraction and proteomic analysis methods previously optimized for formalin-fixed samples. As a result, gastric BFPE protein extracts exhibited poor suitability for 2D-PAGE analysis, whereas over 300 unique proteins could be successfully detected when extracts were subjected to SDS-PAGE followed by LC-MS/MS (GeLC-MS/MS). Among these, several known markers for gastric cancer and normal gastric functionality were identified, indicative of biological and clinical significance of proteomic data mined from BFPE tissues. A quantitative and qualitative comparison of FFPE and BFPE tissue proteomes was also performed, and results are reported. In conclusion, we demonstrated that BFPE specimens can be analyzed by means of a proteomic approach such as GeLC-MS/MS. Although considerable molecular biases and technical constraints exist, BFPE tissue archives can be fruitfully exploited for gathering proteomic data from particularly precious samples.

Characterisation of blood serum peptides can provide valuable information on physiological and pathological processes. However, the analysis of raw serum samples by MS results in the identification of a limited number of peptides. In order to improve sensitivity, many peptide enrichment methods have been proposed during the last ten years. Here, we present a comparison of fractionation methods aimed to simplify analysis of small proteins and peptides in blood serum, one of the most promising sources of putative biomarkers. Specifically, three methods based on ultrafiltration, differential precipitation, and peptide ligand libraries (ProteoMiner) were evaluated for the enrichment of peptides and low molecular weight proteins, as demonstrated by Tricine SDS-PAGE and subsequent LC-MS/MS (GeLC-MS/MS). As a result, differential solubilisation (DS) allowed the identification of the highest number of peptides. Moreover, the DS method enabled also the quantitative comparison of samples, producing fundamental information in biomarker discovery approaches.

The report describes a rapid and simple CE method using LIF detection for the analysis of unsaturated disaccharides obtained from enzymatic depolymerization of plasma chondroitin sulfate (CS) isomers. The disaccharide reducing groups were labeled with 2-aminoacridone (AMAC). The fluorotagged products can be separated by reversed-polarity CE using a sodium acetate buffer, pH 3.8, in the presence of 0.05% methylcellulose. The choice of the appropriate electrophoretic conditions was performed after a deep analysis of the most important parameters affecting analyte separation. In particular, the effect of both run buffer concentration and pH on resolution, efficiency, migration times, and peak area was evaluated. The selected electrophoretic conditions allowed us to separate the CS isomers-derived Delta-disaccharides in less than 12 min, also resolving the nonsulfated disaccharides released from CS isomers from those released from hyaluronan (HA). Moreover, these conditions gave a good reproducibility of both the migration times (CV%, 0.25) and the peak areas (CV%, 1.4). Intra- and interassay CV were 5.37 and 7.23%, respectively, and analytical recovery was about 86%. The applicability of the above method to the quantitative and structural disaccharide analyses of plasma CS isomers was investigated. Data obtained from 44 healthy human subjects were compared with those obtained by a fluorophore-assisted carbohydrate electrophoresis (FACE) reference assay, by using the Passing and Bablok regression and Bland-Altman tests. The developed method could represent a good tool for an ultrasensitive analysis of CS isomers in biological samples from different sources, particularly when samples are available in very low amounts.

Neutrophil extracellular traps (NETs) are structures composed of DNA, histones, and antimicrobial proteins that are released extracellularly by neutrophils and other immune cells as a means for trapping and killing invading pathogens. Here, we describe NET formation in milk and in mammary alveoli of mastitic sheep, and provide a dataset of proteins found in association to these structures. Nucleic acid staining, immunomicroscopy and fluorescent in-situ hybridization of mastitic mammary tissue from sheep infected with Streptococcus uberis demonstrated the presence of extranuclear DNA colocalizing with antimicrobial proteins, histones, and bacteria. Then, proteomic analysis by LTQ-Orbitrap Velos mass spectrometry provided detailed information on protein abundance changes occurring in milk upon infection. As a result, 1095 unique proteins were identified, of which 287 being significantly more abundant in mastitic milk. Upon protein ontology classification, the most represented localization classes for upregulated proteins were the cytoplasmic granule, the nucleus, and the mitochondrion, while function classes were mostly related to immune defence and inflammation pathways. All known NET markers were massively increased, including histones, granule proteases, and antimicrobial proteins. Of note was the detection of protein arginine deiminases (PAD3 and PAD4). These enzymes are responsible for citrullination, the post-translational modification that is known to trigger NET formation by inducing chromatin decondensation and extracellular release of NETs. As a further observation, citrullinated residues were detected by tandem mass spectrometry in histones of samples from mastitic animals. In conclusion, this work provides novel microscopic and proteomic information on NETs formed in vivo in the mammary gland, and reports the most complete database of proteins increased in milk upon bacterial mastitis. Electronic supplementary material The online version of this article (doi:10.1186/s13567-015-0196-x) contains supplementary material, which is available to authorized users.

To date, most metaproteomic studies of the gut microbiota employ stool sample pretreatment methods to enrich for microbial components. However, a specific investigation aimed at assessing if, how, and to what extent this may impact on the final taxonomic and functional results is still lacking. Here, stool replicates were either pretreated by differential centrifugation (DC) or not centrifuged. Protein extracts were then processed by filter-aided sample preparation, single-run LC, and high-resolution MS, and the metaproteomic data were compared by spectral counting. DC led to a higher number of identifications, a significantly richer microbial diversity, as well as to reduced information on the nonmicrobial components (host and food) when compared to not centrifuged. Nevertheless, dramatic differences in the relative abundance of several gut microbial taxa were also observed, including a significant change in the Firmicutes/Bacteroidetes ratio. Furthermore, some important microbial functional categories, including cell surface enzymes, membrane-associated proteins, extracellular proteins, and flagella, were significantly reduced after DC. In conclusion, this work underlines that a critical evaluation is needed when selecting the appropriate stool sample processing protocol in the context of a metaproteomic study, depending on the specific target to which the research is aimed. All MS data have been deposited in the ProteomeXchange with identifier PXD001573 (http://proteomecentral.proteomexchange.org/dataset/PXD001573).

Background The massive characterization of host-associated and environmental microbial communities has represented a real breakthrough in the life sciences in the last years. In this context, metaproteomics specifically enables the transition from assessing the genomic potential to actually measuring the functional expression of a microbiome. However, significant research efforts are still required to develop analysis pipelines optimized for metaproteome characterization. Results This work presents an efficient analytical pipeline for shotgun metaproteomic analysis, combining bead-beating/freeze-thawing for protein extraction, filter-aided sample preparation for cleanup and digestion, and single-run liquid chromatography-tandem mass spectrometry for peptide separation and identification. The overall procedure is more time-effective and less labor-intensive when compared to state-of-the-art metaproteomic techniques. The pipeline was first evaluated using mock microbial mixtures containing different types of bacteria and yeasts, enabling the identification of up to over 15,000 non-redundant peptide sequences per run with a linear dynamic range from 104 to 108 colony-forming units. The pipeline was then applied to the mouse fecal metaproteome, leading to the overall identification of over 13,000 non-redundant microbial peptides with a false discovery rate of

In this study, the proteome profile of European sea bass (Dicentrarchus labrax) muscle was analyzed using two-dimensional electrophoresis (2-DE) and tandem mass spectrometry with the aim of providing a more detailed characterization of its specific protein expression profile. A highly populated and well-resolved 2-DE map of the sea bass muscle tissue was generated, and the corresponding protein identity was provided for a total of 49 abundant protein spots. Upon Ingenuity Pathway Analysis, the proteins mapped in the sea bass muscle profile were mostly related to glycolysis and to the muscle myofibril structure, together with other biological activities crucial to fish muscle metabolism and contraction, and therefore to fish locomotor performance. The data presented in this work provide important and novel information on the sea bass muscle tissue-specific protein expression, which can be useful for future studies aimed to improve seafood traceability, food safety/risk management and authentication analysis. This work is also important for understanding the proteome map of the sea bass toward establishing the animal as a potential model for muscular studies.

Bouin's solution has been used for over a century as a common fixative in several pathology laboratories worldwide. Therefore, a considerable number of Bouin-fixed paraffin-embedded (BFPE) tumor samples of various origin are available in hospital repositories as a powerful information mine for clinical investigations. To date, however, such archived tissues have not been subjected to a systematic study aimed to evaluate their potential use in proteomics. In this report, we investigated whether archival BFPE tissue specimens could be exploited for proteomic studies, upon application of protein extraction and proteomic analysis methods previously optimized for formalin-fixed samples. As a result, gastric BFPE protein extracts exhibited poor suitability for 2D-PAGE analysis, whereas over 300 unique proteins could be successfully detected when extracts were subjected to SDS-PAGE followed by LC-MS/MS (GeLC-MS/MS). Among these, several known markers for gastric cancer and normal gastric functionality were identified, indicative of biological and clinical significance of proteomic data mined from BFPE tissues. A quantitative and qualitative comparison of FFPE and BFPE tissue proteomes was also performed, and results are reported. In conclusion, we demonstrated that BFPE specimens can be analyzed by means of a proteomic approach such as GeLC-MS/MS. Although considerable molecular biases and technical constraints exist, BFPE tissue archives can be fruitfully exploited for gathering proteomic data from particularly precious samples.

Two-dimensional difference gel electrophoresis (2D DIGE) was applied to investigate the impact of slaughtering on the postmortem integrity of muscle tissue proteins in European sea bass (Dicentrarchus labrax). Three different slaughtering techniques were evaluated: asphyxia in air (AA), asphyxia in ice (AI), and spinal cord severance (SCS). Principal components analysis (PCA) revealed a significant divergence of SCS samples, whereas AA and AI samples, although grouped separately, were less divergent and could be included in a single asphyxia cluster. In terms of single proteins, the most significant impact was seen on nucleoside diphosphate kinase B, which was consistently less affected when fish were slaughtered by SCS as compared to asphyxia. Integrity of the sarcomeric proteins myosin heavy chain and myosin binding protein C and of the cytosolic proteins fructose biphosphate aldolase, glyceraldehyde 3-phosphate dehydrogenase, and enolase 1 was also better preserved upon SCS slaughtering. Most interestingly, the influence on muscle protein integrity could be detected since the early postmortem phase. In conclusion, slaughtering by SCS preserves protein integrity better than death by asphyxia, either in ice or in air. Both asphyxia conditions are comparably more adverse than SCS to muscle protein integrity, although a general trend favoring AI over AA is observed.

Several studies have evidenced variations in plasma glycosaminoglycans content in physiological and pathological conditions. In normal human plasma GAGs are present mainly as undersulfated chondroitin sulfate (CS). The aim of the present study was to evaluate possible correlations between plasma CS level/structure and the presence/typology of carotid atherosclerotic lesion. Plasma CS was purified from 46 control subjects and 47 patients undergoing carotid endarterectomy showing either a soft or a hard plaque. The concentration and structural characteristics of plasma CS were assessed by capillary electrophoresis of constituent unsaturated fluorophore-labeled disaccharides. Results showed that the concentration of total CS isomers was increased by 21.4% (P<0.01) in plasma of patients, due to a significant increase of undersulfated CS. Consequently, in patients the plasma CS charge density was significantly reduced with respect to that of controls. After sorting for plaque typology, we found that patients with soft plaques and those with hard ones differently contribute to the observed changes. In plasma from patients with soft plaques, the increase in CS content was not associated with modifications of its sulfation pattern. On the contrary, the presence of hard plaques was associated with CS sulfation pattern modifications in presence of quite normal total CS isomers levels. These results suggest that the plasma CS content and structure could be related to the presence and the typology of atherosclerotic plaque and could provide a useful diagnostic tool, as well as information on the molecular mechanisms responsible for plaque instability.

Storage conditions are known to be important for postmortem deterioration of fish muscle, and temperature is one of the factors with the strongest impact on this process. In order to shed light on the influence of temperature on the status of sea bass (Dicentrarchus labrax) muscle proteins during postmortem storage, a 2-D DIGE and mass spectrometry study was performed on fish kept at either 1 or 18°C for 5 days. As expected, the greatest alterations in sea bass filet protein composition were observed upon postmortem storage at 18°C, with distinct changes appearing in the 2-D protein profile after 5 days of storage at this temperature. In particular, degradation of the myofibrillar protein myosin heavy chain and of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, among the most abundant muscle proteins, could be clearly observed upon storage at higher temperatures. Although to a lesser extent, however, several proteins were observed to vary in abundance also upon storage for 5 days at 1°C. In particular, one of the most interesting observations was the rapid and significant decrease in the abundance of nucleoside diphosphate kinase B and phosphoglycerate mutase 2, which was observed also at low storage temperatures and appeared to be temperature-independent. The results of this study offer new knowledge on changes occurring in sea bass muscle proteins during postmortem storage at different temperatures and provide indications on protein degradation trends that might be useful for monitoring freshness of fish and quality of storage conditions.

Milk fat globules (MFGs) are vesicles released in milk as fat droplets surrounded by the endoplasmic reticulum and apical cell membranes. During formation and apocrine secretion by lactocytes, various amounts of cytoplasmic crescents remain trapped within the released vesicle, making MFGs a natural sampling mechanism of the lactating cell contents. With the aim of investigating the events occurring in the mammary epithelium during bacterial infection, the MFG proteome was characterized by two-dimensional difference gel electrophoresis (2-D DIGE), SDS-PAGE followed by shotgun liquid chromatography-tandem mass spectrometry (GeLC-MS/MS), label-free quantification by the normalized spectral abundance factor (NSAF) approach, Western blotting, and pathway analysis, using sheep naturally infected by Mycoplasma agalactiae . A number of protein classes were found to increase in MFGs upon infection, including proteins involved in inflammation and host defense, cortical cytoskeleton proteins, heat shock proteins, and proteins related to oxidative stress. Conversely, a strikingly lower abundance was observed for proteins devoted to MFG metabolism and secretion. To our knowledge, this is the first report describing proteomic changes occurring in MFGs during sheep infectious mastitis. The results presented here offer new insights into the in vivo response of mammary epithelial cells to bacterial infection and open the way to the discovery of protein biomarkers for monitoring clinical and subclinical mastitis.

Background The growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research. Direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) or filter-aided sample preparation (FASP) are the most common workflows for shotgun analysis of FFPE samples, but a critical comparison of the different methods is currently lacking. Experimental design DT, FASP and ISD workflows were compared by subjecting to the same label-free quantitative approach three independent technical replicates of each method applied to FFPE liver tissue. Data were evaluated in terms of method reproducibility and protein/peptide distribution according to localization, MW, pI and hydrophobicity. Results DT showed lower reproducibility, good preservation of high-MW proteins, a general bias towards hydrophilic and acidic proteins, much lower keratin contamination, as well as higher abundance of non-tryptic peptides. Conversely, FASP and ISD proteomes were depleted in high-MW proteins and enriched in hydrophobic and membrane proteins; FASP provided higher identification yields, while ISD exhibited higher reproducibility. Conclusions These results highlight that diverse sample preparation strategies provide significantly different proteomic information, and present typical biases that should be taken into account when dealing with FFPE samples. When a sufficient amount of tissue is available, the complementary use of different methods is suggested to increase proteome coverage and depth.

We present a human gut metaproteomic dataset deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD001573. Ten aliquots of a single stool sample collected from a healthy human volunteer were either pretreated by differential centrifugation (DC; N=5) or not centrifuged (NC; N=5). Protein extracts were then processed by filter-aided sample preparation, single-run liquid chromatography and high-resolution mass spectrometry, and peptide identification was carried out using Sequest-HT as search engine within the Proteome Discoverer informatic platform. The dataset described here is also related to the research article entitled “Enrichment or depletion? The impact of stool pretreatment on metaproteomic characterization of the human gut microbiota” published in Proteomics (Tanca et al., 2015), [1].

Here we present a dataset generated using formalin-fixed paraffin-embedded archival samples from two rare lung neuroendocrine tumor subtypes (namely, two atypical carcinoids, ACs, and two large-cell neuroendocrine carcinomas, LCNECs). Samples were subjected to a shotgun proteomics pipeline, comprising full-length protein extraction, SDS removal through spin columns, in solution trypsin digestion, long gradient liquid chromatography peptide separation and LTQ-Orbitrap mass spectrometry analysis. A total of 1260 and 2436 proteins were identified in the AC and LCNEC samples, respectively, with FDR

Mastitis due to intramammary infections is one of the most detrimental diseases in dairy sheep farming, representing a major cause of reduced milk productions and quality losses. In particular, subclinical mastitis presents significant detection and control problems, and the availability of tools enabling its timely, sensitive, and specific detection is therefore crucial. We have previously demonstrated that cathelicidins, small proteins implicated in the innate immune defense of the host, are specifically released in milk of mastitic animals by both epithelial cells and neutrophils. Here, we describe the development of an ELISA for milk cathelicidin and assess its value against somatic cell counts (SCC) and bacteriological culture for detection of ewe mastitis. Evaluation of the cathelicidin ELISA was carried out on 705 half-udder milk samples from 3 sheep flocks enrolled in a project for improvement of mammary health. Cathelicidin was detected in 35.3% of milk samples (249/705), and its amount increased with rising SCC values. The cathelicidin-negative (n=456) and cathelicidin-positive (n=249) sample groups showed a clear separation in relation to SCC, with median values of 149,500 and 3,300,000 cells/mL, respectively. Upon bacteriological culture, 20.6% (145/705) of the milk samples showed microbial growth, with coagulase-negative staphylococci being by far the most frequent finding. A significant proportion of all bacteriologically positive milk samples were positive for cathelicidin (110/145, 75.9%). Given the lack of a reliable gold standard for defining the true disease status, sensitivity (Se) and specificity (Sp) of the cathelicidin ELISA were assessed by latent class analysis against 2 SCC thresholds and against bacteriological culture results. At an SCC threshold of 500,000 cells/mL, Se and Sp were 92.3 and 92.3% for cathelicidin ELISA, 89.0 and 94.9% for SCC, and 39.4 and 93.6% for bacteriological culture, respectively. At an SCC threshold of 1,000,000 cells/mL, Se and Sp were 93.3 and 91.9% for cathelicidin ELISA, 80.0 and 97.1% for SCC, and 39.4 and 93.5% for bacteriology, respectively. In view of the results obtained in this study, the measurement of cathelicidin in milk by ELISA can provide added Se while maintaining a high Sp and may therefore improve detection of subclinical mastitis.

Mastitis due to intramammary infection is one of the most economically relevant diseases in dairy cows, causing reductions in milk quality and quantity. Currently, mastitis monitoring is based on somatic cell count (SCC) and bacteriologic culture (BC) of milk. Nevertheless, inflammation-specific protein markers might provide more sensitive and reliable assays, enabling immunoassay-based screening strategies. Cathelicidin is an inflammatory protein released in milk that has recently demonstrated fair reliability and diagnostic potential for ewe mastitis. To assess its performance in cows, 531 quarter milk samples from 2 herds were tested using cathelicidin ELISA, SCC, and BC. We found that 29.0% of samples were positive for cathelicidin, 18.8% had SCC >200,000 cells/mL, and 13.7% were BC-positive. Cathelicidin showed a strong positive correlation with SCC as demonstrated by receiver operating characteristics curve analysis and by the clustering of cathelicidin-negative and cathelicidin-positive samples in association with low and high SCC values, respectively. For evaluating the diagnostic performance of a novel test, BC cannot be considered a reliable gold standard for true disease status because of its known limitations. Therefore, we assessed the sensitivity (Se) and specificity (Sp) of the milk cathelicidin ELISA using a latent class analysis approach together with BC and SCC by considering different diagnostic thresholds to identify the preferred Se/Sp combination. We modeled conditional dependence of cathelicidin and SCC to account for their close association. The cathelicidin ELISA showed higher Se than SCC and BC for almost all threshold combinations. In fact, at the best-performing threshold combination, the Se of cathelicidin was 80.6%, 6.2 percentage points higher than that of SCC >200,000 cells/mL (74.4%) and similar to that of SCC >100,000 cells/mL (80.2%). Most importantly, this Se was obtained with a loss in Sp of only 1.4 percentage points compared with SCC >200,000 cells/mL (94.9% Sp for cathelicidin vs. 96.3% for SCC >200,000). The limited Se of BC (38.8%) was also confirmed in this study, and BC showed a slightly lower Sp than both cathelicidin and SCC for most of threshold combinations. This study confirmed that cathelicidin is released in the milk of cows with mastitis and that its presence is highly correlated with SCC. The measurement of cathelicidin by ELISA may hold significant potential for improving the sensitivity of mastitis detection in dairy cows while maintaining high specificity.