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Inulin is a natural polysaccharide classified as a soluble fiber with demonstrated prebiotic activity. Prebiotics can reduce intestinal and systemic inflammation through modulation of the gut microflora and their metabolites. Additionally, extensive research is illuminating the role of macrophages in the interaction between gut microbiota and many systemic inflammatory diseases. In this study, the anti-inflammatory properties of inulin were evaluated using a murine macrophage cell model (RAW 264.7) of inflammation, and the immunomodulatory mechanism was investigated using omics technologies. The cells underwent comprehensive transcriptomic and proteomic analyses to identify the mechanisms responsible for the observed anti-inflammatory phenotype. Functional analyses of these omics results revealed two potential mechanisms that may lead to an overall reduction in cytokine and chemokine transcription: the inhibition of the NF-κB signaling pathway, leading to the downregulation of proinflammatory factors such as COX2, and the promotion of the phase II defense protein Hmox1 via the Nrf2 pathway. This study provides promising targets for research on immune modulation by dietary fibers and offers new strategies for the design of functional ingredients, foods, and nutraceutical products, which could ultimately lead to personalized nutrition and improved consumer health.

Therapy with anti-tumor necrosis factor (TNF) has dramatically changed the natural history of Crohn’s disease (CD). However, these drugs are not without adverse events, and up to 40% of patients could lose efficacy in the long term. We aimed to identify reliable markers of response to anti-TNF drugs in patients with CD. A consecutive cohort of 113 anti-TNF naive patients with CD was stratified according to clinical response as short-term remission (STR) or non-STR (NSTR) at 12 weeks of treatment. We compared the protein expression profiles of plasma samples in a subset of patients from both groups prior to anti-TNF therapy by SWATH proteomics. We identified 18 differentially expressed proteins (p ≤ 0.01, fold change ≥ 2.4) involved in the organization of the cytoskeleton and cell junction, hemostasis/platelet function, carbohydrate metabolism, and immune response as candidate biomarkers of STR. Among them, vinculin was one of the most deregulated proteins (p < 0.001), whose differential expression was confirmed by ELISA (p = 0.054). In the multivariate analysis, plasma vinculin levels along with basal CD Activity Index, corticosteroids induction, and bowel resection were factors predicting NSTR.

The relationship between the functionality and composition of high-density lipoproteins (HDL) is yet not fully studied, and little is known about the influence of the diet in HDL proteome. Therefore, the aim of this research was to elucidate the HDL proteome associated to postprandial hyperlipidemia. Male volunteers were recruited for an interventional study with high fatty acid-based meals. Blood samples were collected before the intake (baseline), and 2-3 (postprandial peak) and 5-6 (postprandial post peak) hours later. HDL were purified and the protein composition was quantified by LC-MS/MS. Statistical analysis was performed by lineal models (amica) and by ANOVA and multi-t-test of the different conditions (MetaboAnalyst). Additionally, a clustering of the expression profiles of each protein was done with coseq R package (RStudio). Initially, 320 proteins were identified but only 119 remained after the filtering. APOM, APOE, APOB, and APOA2, proteins previously identified in the HDL proteome, were the only proteins with a statistically significant altered expression in postprandial hyperlipidemia when compared to baseline (p values0.05 and logFC1). In conclusion, we have been able to describe several behaviors of the whole HDL proteome during the postprandial hyperlipidemic metabolism.

This article belongs to the Special Issue Towards Precision Prognostication and Personalized Therapeutics through Proteomics., The high mortality, the presence of an initial asymptomatic stage and the fact that diagnosis in early stages reduces mortality justify the implementation of screening programs in the populations at risk of lung cancer. It is imperative to develop less aggressive methods that can complement existing diagnosis technologies. In this study, we aimed to identify lung cancer protein biomarkers and pathways affected in sputum samples, using the recently developed diaPASEF mass spectrometry (MS) acquisition mode. The sputum proteome of lung cancer cases and controls was analyzed through nano-HPLC–MS using the diaPASEF mode. For functional analysis, the results from differential expression analysis were further analyzed in the STRING platform, and feature selection was performed using sparse partial least squares discriminant analysis (sPLS-DA). Our results showed an activation of inflammation, with an alteration of pathways and processes related to acute-phase, complement, and immune responses. The resulting sPLS-DA model separated between case and control groups with high levels of sensitivity and specificity. In conclusion, we showed how new-generation proteomics can be used to detect potential biomarkers in sputum samples, and ultimately to discriminate patients from controls and even to help to differentiate between different cancer subtypes., This work was supported by the Proyectos de Investigación en Salud 2020 programme of the Consejería de Salud y Familias, Junta de Andalucía, with grant number PI-0015-2020.

The disease known as coronavirus disease 19 (COVID-19), potentially caused by an outbreak of the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in Wuhan, China, has hit the world hard, and has led to an unprecedent health and economic crisis. In order to develop treatment options able to stop or ameliorate SARS-CoV-2 effects, we need to understand the biology of the virus inside cells, but this kind of studies are still scarce. A recent study investigated translatome and proteome host cell changes induced in vitro by SARS-CoV-2. In the present study, we use the publicly available proteomics data from this study to re-analyze the mechanisms altered by the virus infection by impact pathways analysis and network analysis. Proteins linked to inflammatory response, but also proteins related to chromosome segregation during mitosis, were found to be regulated. The up-regulation of the inflammatory-related proteins observed could be linked to the propagation of inflammatory reaction and lung injury that is observed in advanced stages of COVID-19 patients.

9 pages, 4 figures, 2 tables.-- Under a Creative Commons license, This review illustrates how the use of the latest omics technologies is being applied to investigate the effect of diets, foods or food components on health/disease status. Bioactive food components alter gene expression and protein levels and functions, leading to various beneficial effects on human health. Gene variants and expression, epigenetic regulation, protein levels and post-translational modifications, as well as metabolites, can be analyzed and integrated globally to obtain functional information. Foodomics, integrating the study of food and nutrition with omics technologies, allows the investigation of cellular processes, functional mechanisms and molecules involved, as well as the definition of targets for bioactive compounds useful for the development of nutritional intervention strategies, and the discovery of biomarkers linking nutrition and health. In addition, the main limitations of the use of these approaches are discussed, as well as the trends that will be applied in the area in the near future, providing a broad and updated view

Data-independent acquisition (DIA) methods have gained great popularity in bottom-up quantitative proteomics, as they overcome the irreproducibility and under-sampling limitations of data-dependent acquisition (DDA). diaPASEF, recently developed for the timsTOF Pro mass spectrometers, has brought improvements to DIA, providing additional ion separation (in the ion mobility dimension) and increasing sensitivity. Several studies have benchmarked different workflows for DIA quantitative proteomics, but mostly using instruments from Sciex and Thermo, and therefore, the results are not extrapolable to diaPASEF data. In this work, using a real-life sample set like the one that can be found in any proteomics experiment, we compared the results of analyzing PASEF data with different combinations of library-based and library-free analysis, combining the tools of the FragPipe suite, DIA-NN and including MS1-level LFQ with DDA-PASEF data, and also comparing with the workflows possible in Spectronaut. We verified that library-independent workflows, not so efficient not so long ago, have greatly improved in the recent versions of the software tools, and now perform as well or even better than librarybased ones. We report here information so that the user who is going to conduct a relative quantitative proteomics study using a timsTOF Pro mass spectrometer can make an informed decision on how to acquire (diaPASEF for DIA analysis, or DDA-PASEF for MS1-level LFQ) the samples, and what can be expected depending on the data analysis tool used, among the different alternatives offered by the recently optimized tools for TIMS-PASEF data analysis.

Adulteration and mislabeling of food products and the commercial fraud derived, either intentionally or not, is a global source of economic fraud to consumers but also to all stakeholders involved in food production and distribution. Legislation has been enforced all over the world aimed at guaranteeing the authenticity of the food products all along the distribution chain, thereby avoiding food fraud and adulteration. Accordingly, there is a growing need for new analytical methods able to verify that all the ingredients included in a foodstuff match the qualities claimed by the manufacturer or distributor. In this sense, the improved performance of most recent DNA-based tools in term of sensitivity, multiplexing ability, high-throughput, and relatively low-cost give them a game-changing role in food-authenticity-related topics. Here, we provide a thorough and updated vision on the recently reported approaches that are applying these DNA-based tools to assess the authenticity of food components and products.

Classical and culture-based methods for the identification and characterization of the biochemical properties of microorganisms are slow and labor-intensive. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) has been used for the analysis of bacterial pathogen strain-specific diagnostic peptides allowing the characterization of bacterial strains.Here, we describe the analysis of tryptic digestion peptides by LC-ESI-MS/MS to search for specific biomarkers useful for the rapid identification of, on the one hand, the bacterial species and, on the other hand, the physiological and biochemical characteristics such as the expression of virulence factors, including toxins, immune-modulatory factors, and exoenzymes.

Classical and culture-based methods for the identification and characterization of the biochemical properties of microorganisms are slow and labor-intensive. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) has been used for the analysis of bacterial pathogen strain-specific diagnostic peptides allowing the characterization of bacterial strains.Here, we describe the analysis of tryptic digestion peptides by LC-ESI-MS/MS to search for specific biomarkers useful for the rapid identification of, on the one hand, the bacterial species and, on the other hand, the physiological and biochemical characteristics such as the expression of virulence factors, including toxins, immune-modulatory factors, and exoenzymes.

α-Linolenic acid (ALA, 18:3n-3) and γ-gamma linolenic acid (GLA, 18:3n-6) are polyunsaturated fatty acids (PUFA) that improve the human health. The present study focused on testing the in vitro antitumor actions of pure ALA and GLA on the HT-29 human colorectal cancer cell line. Cell viability was checked by MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, cell membrane damage by the lactate dehydrogenase assay, apoptosis was tested by both caspase-3 activity trial and transmission electron microscopy images, and protein composition was analyzed by quantitative proteomics analysis. MTT test revealed IC50 values of 230 and 255 μM for ALA and GLA, respectively, at 72 h. After 24 h of incubation, both ALA and GLA induced apoptosis on HT-29 colorectal cancer cells according to the caspase-3 assay and microscopy images. SWATH/MS analysis evidenced that ALA significantly affected the mitochondrial protein import pathway and the citric acid cycle pathway, while GLA did not significantly affect any particular pathway. In summary, both ALA and GLA showed concentration-dependent inhibitory effects on HT-29 cells viability and induced cell death by apoptosis. ALA significantly affected cellular pathways, while GLA does not have specific actions on either pathway. Both n-3 and n-6 C18 PUFA are bioactive food components useful in the colorectal cancer prevention.

Food adulteration, mislabeling and fraud, either unintentional or deliberate, is a worldwide and growing concern. Current challenges associated with food authentication comprise many topics: geographic origin, breed/variety identification, production method, feeding regime, technological processing, undeclared components, detection of genetically modified organisms and species substitution are the most common frauds. Accurate and reliable analytical methods are needed to monitor and control food authenticity and to guarantee correct and accurate labeling of foodstuffs, assuring that the components included in a food product are of the nature and quality declared by the seller. Omics-based massive molecular tools can help to circumvent limitations of traditional methodologies, and therefore genomics, proteomics and metabolomics-based methods are being developed for the authentication of a wide range of food commodities. In this review, we provide an updated, comprehensive and balanced overview of the recent studies (2015–2018) that have applied these omics-based technologies for the authentication of food features.

Two polyunsaturated fatty acids, docosahexaenoic acid (DHA) and arachidonic acid (ARA), as well as derivatives, such as eicosanoids, regulate different activities, affecting transcription factors and, therefore, DNA transcription, being a critical step for the functioning of fatty-acid-derived signaling. This work has attempted to determine the in vitro anticancer activities of these molecules linked to the gene transcription regulation of HT-29 colorectal cancer cells. We applied the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test along with lactate dehydrogenase and caspase-3 assays; proteome changes were assessed by "sequential windowed acquisition of all theoretical mass spectra" quantitative proteomics, followed by pathway analysis, to determine the affected molecular mechanisms. In all assays, DHA inhibited cell proliferation of HT-29 cells to a higher extent than ARA and acted primarily by downregulating proteasome particles, while ARA presented a dramatic effect on all six DNA replication helicase particles. The results indicated that both DHA and ARA are potential chemopreventive agent candidates.

Colorectal cancer (CRC) is one of the most common and mortal types of cancer. There is increasing evidence that some polyunsaturated fatty acids (PUFAs) exercise specific inhibitory actions on cancer cells through different mechanisms, as a previous study on CRC cells demonstrated for two very long-chain PUFA. These were docosahexaenoic acid (DHA, 22:6n3) and arachidonic acid (ARA, 20:4n6) in the free fatty acid (FFA) form. In this work, similar design and technology have been used to investigate the actions of both DHA and ARA as monoacylglycerol (MAG) molecules, and results have been compared with those obtained using the corresponding FFA. Cell assays revealed that ARA- and DHA-MAG exercised dose- and time-dependent antiproliferative actions, with DHA-MAG acting on cancer cells more efficiently than ARA-MAG. Sequential window acquisition of all theoretical mass spectra (SWATH) &ndash, mass spectrometry massive quantitative proteomics, validated by parallel reaction monitoring and followed by pathway analysis, revealed that DHA-MAG had a massive effect in the proteasome complex, while the ARA-MAG main effect was related to DNA replication. Prostaglandin synthesis also resulted as inhibited by DHA-MAG. Results clearly demonstrated the ability of both ARA- and DHA-MAG to induce cell death in colon cancer cells, which suggests a direct relationship between chemical structure and antitumoral actions.

Newly emerged proteomic methodologies, particularly data‐independent acquisition (DIA) analysis–related approaches, would improve current gene expression–based classifications of colorectal cancer (CRC). Therefore, this study was aimed to identify protein expression signatures using SWATH‐MS DIA and targeted data extraction, to aid in the classification of molecular subtypes of CRC and advance in the diagnosis and development of new drugs. For this purpose, 40 human CRC samples and 7 samples of healthy tissue were subjected to proteomic and bioinformatic analysis. The proteomic analysis identified three different molecular CRC subtypes: P1, P2 and P3. Significantly, P3 subtype showed high agreement with the mesenchymal/stem‐like subtype defined by gene expression signatures and characterized by poor prognosis and survival. The P3 subtype was characterized by decreased expression of ribosomal proteins, the spliceosome, and histone deacetylase 2, as well as increased expression of osteopontin, SERPINA 1 and SERPINA 3, and proteins involved in wound healing, acute inflammation and complement pathway. This was also confirmed by immunodetection and gene expression analyses. Our results show that these tumours are characterized by altered expression of proteins involved in biological processes associated with immune evasion and metastasis, suggesting new therapeutic options in the treatment of this aggressive type of CRC.

Consumers have the right to know what is in the food they are eating. Accordingly, European and global food regulations require that the provenance of the food can be guaranteed from farm to fork. Many different instrumental techniques have been proposed for food authentication. Although traditional methods are still being used, new approaches such as genomics, proteomics, and metabolomics are helping to complement existing methodologies for verifying the claims made about certain food products. During the last decade, proteomics (the large-scale analysis of proteins in a particular biological system at a particular time) has been applied to different research areas within food technology. Since proteins can be used as markers for many properties of a food, even indicating processes to which the food has been subjected, they can provide further evidence of the foods labeling claim. This review is a comprehensive and updated overview of the applications, drawbacks, advantages, and challenges of proteomics for food authentication in the assessment of the foods compliance with labeling regulations and policies. Significance This review paper provides a comprehensive and critical overview of the application of proteomics approaches to determine the authenticity of several food products updating the performances and current limitations of the applied techniques in both laboratory and industrial environments.

Galiellalactone (GL) is a fungal metabolite that presents antitumor and antiinflammatory activities in vitro and in vivo. Previous studies have shown that GL targets NF-κB and STAT3 pathways and induces G2/M cell cycle arrest in androgen-insensitive prostate cancer cells. In this study, we show that GL-induced cell cycle arrest is independent of the NF-κB and STAT3 pathways in DU145 and PC-3 cells, and also that GL did not affect cell cycling in androgen-sensitive prostate cancer cells such as LNCaP and 22Rv1 cells. In addition, we showed confluence resistance to GL in DU145 cells. Using a SWATH proteomic approach we identified a down-regulation of Nucleolar and spindle associated protein 1 (NUSAP1) under DU145 confluence. Moreover the expression of NUSAP1 in LNCaP cells is low compared to DU145 cells. The inhibition of NUSAP1 by siRNAs induced resistance to GL in DU145 cells, suggesting that NUSAP1 may be a target for GL and could be useful as a biomarker for the responsiveness of the antitumor activity of GL. Altogether, our finding shed light to the potential of GL to be developed as a novel treatment of castration resistance prostate cancer.

This work has been financially supported by: the Plan Nacional de IþDþI 2013–2016/FEDER (PI15/00892 to M.F.F. and A.F.F.; RTC2015-3393-1 to A.F.F.); the ISCIII-Subdirección General de Evaluación y Fomento de la Investigación, and the Plan Nacional de IþDþI 2008–2011/FEDER (CP11/00131 to A.F.F.); IUOPA (to G.F.B. and M.S); the Fundación Científica de la AECC (to R.G.U.); the Fundación Ramón Areces (to M.F.F); FICYT (to E.G.T., M.G.G. and A.C.); and the Asturias Regional Government (GRUPIN14-052 to M.F.F.). Work in P.M. laboratory is supported by the European Research Council (CoG-2014-646903), the Spanish Ministry of Economy-Competitiveness (SAF-SAF2013-43065), the Obra Social La Caixa-Fundació Josep Carreras, and the Generalitat de Catalunya. P.M. is an investigator in the Spanish Cell Therapy cooperative network (TERCEL). The IUOPA is supported by the Obra Social Cajastur-Liberbank, Spain., Fernandez, A.F., Bayón, G.F., Sierra, M.I., Urdinguio, R.G., Toraño, E.G., García, M.G., Carella, A., López, V., Santamarina, P., Pérez, R.F., Belmonte, T., Tejedor, J.R., Cobo, I., Menendez, P., Mangas, C., Ferrero, C., Rodrigo, L., Astudillo, A., Ortea, I., Díaz, S.C., Rodríguez-Gonzalez, P., Alonso, J.I.G., Mollejo, M., Meléndez, B., Domínguez, G., Bonilla, F., Fraga, M.F.

Aberrant DNA hypermethylation is a hallmark of cancer although the underlying molecular mechanisms are still poorly understood. To study the possible role of 5-hydroxymethylcytosine (5hmC) in this process we analyzed the global and locus-specific genome-wide levels of 5hmC in primary samples from 54 gliomas and 72 colorectal cancer patients. Levels of 5hmC in colorectal cancer were very low and no consistent changes were detected between control tissues and tumors. As expected, levels of 5hmC in non-tumoral brain samples were high and significantly reduced at the 49,601 CpG sites in gliomas. Strikingly, hypo-hydroxymethylation at 4,627 (9.3%) of these CpG sites was associated with aberrant DNA hypermethylation. The DNA regions containing these CpG sites were enriched in H3K4me2, and presented a different genuine chromatin signature to that characteristic of the genes classically aberrantly hypermethylated in cancer. We conclude that this data identifies a novel 5hmC-dependent non-canonical class of aberrant DNA hypermethylation in glioma.

A typical clinical manifestation of growth hormone deficiency (GHD) is a short stature resulting from delayed growth, but GHD affects bone health, cardiovascular function and metabolic profile and therefore quality of life. Although early GH treatment during childhood has been shown to improve outcomes, no single biochemical parameter is currently available for the accurate diagnosis of GHD in children. There is hence a need for non-invasive biomarkers. In this study, the relative abundance of serum proteins from GHD children and healthy controls was measured by next-generation proteomics SWATH-MS technology. The data generated was analysed by machine-learning feature-selection algorithms in order to discover the minimum number of protein biomarkers that best discriminate between both groups. The analysis of serum proteins by a SWATH-MS approach yielded a useful method for discovering potential biomarkers of GHD in children. A total of 263 proteins were confidently detected and quantified in each sample. Pathway analysis indicated an effect on tissue/organ structure and morphogenesis. The top ten serum protein biomarker candidates were identified after applying feature-selection data analysis. The combination of three proteins - apolipoprotein A-IV, complement factor H-related protein 4 and platelet basic protein - demonstrated the best classification performance for our data. In addition, the apolipoprotein group resulted in strong over-representation, thus highlighting these proteins as an additional promising biomarker panel.Currently there is no single biochemical parameter available for the accurate diagnosis of growth hormone (GH) deficiency (GHD) in children. Simple GH measurements are not an option: because GH is released in a pulsatile action, its blood levels fluctuate throughout the day and remain nearly undetectable for most of that time. This makes measurements of GH in a single blood sample useless for assessing GH deficiency. Actually, the diagnosis of GHD includes a combination of direct and indirect non-accurate measurements, such as taking several body measurements, testing GH levels in multiple blood samples after provocative tests (GH peak7.3ng/mL, using radioimmunoassay), and conducting magnetic resonance imaging (MRI), among others. Therefore, there is a need for simple, non-invasive, accurate and cost-effective biomarkers. Here we report a case-control study, where relative abundance of serum proteins were measured by next-generation proteomics SWATH-MS technology in 15 GHD children and 15healthy controls matched by age, sex, and not receiving any treatment. Data generated was analysed by machine learning feature selection algorithms. 263 proteins could be confidently detected and quantified on each sample. The top 10 serum protein biomarker candidates could be identified after applying a feature selection data analysis. The combination of three proteins, apolipoprotein A-IV, complement factor H-related protein 4 and platelet basic protein, showed the best classification performance for our data. In addition, the fact that the pathway and GO analysis we performed pointed to the apolipoproteins as over-represented highlights this protein group as an additional promising biomarker panel for the diagnosis of GHD and for treatment evaluation.

8 pages, 3 figures, This review is a critical overview of advances in proteomics applied in food technology, which may be classified into two main topics: (i) authentication of food components as a tool to comply with food-labeling regulations and policies; and, (ii) food-technology research, mainly for the development of fast, reliable methods to detect and to identify spoilage and/or pathogenic microorganisms in food and for the study of changes in food components as a consequence of food processing.

12 pages, Food components are regularly adulterated, either deliberately or unintentionally, and this may lead to substitutions of a species by a similar but lower-quality counterpart, mislabeling of products, and therefore to commercial fraud. The authentication of food products is a request from consumers and industry, and accordingly global food regulations have been implemented in order to fight against food adulteration and mislabeling. Many different instrumental techniques have been proposed for species authentication in foodstuffs as tools to comply with food labeling regulations and policies. Although traditional methods such as liquid and gas chromatography, nuclear magnetic resonance, infrared and fluorescence spectroscopy, capillary electrophoresis, and more recently, immunological technologies (e.g., ELISA) and DNA approaches are still being used, they are now being complemented by proteomics-based approaches employing electrophoretic and mass spectrometry methodologies. In this chapter, we offer a comprehensive and updated overview of the proteomics-related studies that have been applied to date for the species authentication in seafood products

Despite the maturity reached by targeted proteomic strategies, reliable and standardized protocols are urgently needed to enhance reproducibility among different laboratories and analytical platforms, facilitating a more widespread use in biomedical research. To achieve this goal, the use of dimensionless relative retention times (iRT), defined on the basis of peptide standard retention times (RT), has lately emerged as a powerful tool. The robustness, reproducibility and utility of this strategy were examined for the first time in a multicentric setting, involving 28 laboratories that included 24 of the Spanish network of proteomics laboratories (ProteoRed-ISCIII). According to the results obtained in this study, dimensionless retention time values (iRTs) demonstrated to be a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups both intra- and inter-laboratories. iRT values also showed very low variability over long time periods. Furthermore, parallel quantitative analyses showed a high reproducibility despite the variety of experimental strategies used, either MRM (multiple reaction monitoring) or pseudoMRM, and the diversity of analytical platforms employed. [Biological significance]: From the very beginning of proteomics as an analytical science there has been a growing interest in developing standardized methods and experimental procedures in order to ensure the highest quality and reproducibility of the results. In this regard, the recent (2012) introduction of the dimensionless retention time concept has been a significant advance. In our multicentric (28 laboratories) study we explore the usefulness of this concept in the context of a targeted proteomics experiment, demonstrating that dimensionless retention time values is a useful tool for transferring and sharing peptide retention times across different chromatographic set-ups., All laboratories from Spain are members of ProteoRed (Plataforma de Recursos Biomoleculares y Bioinformáticos) and are supported bygrant PT13/0001 funded by Instituto de Salud Carlos III (ISCIII) andFEDER

Biologics such as TNF antagonists are a new class of drugs that have greatly improved Rheumatoid Arthritis (RA) treatment. However, for unknown reasons, individual patients with RA respond to one of these drugs but not to others even those targeting the same molecule. Methods to predict response are sorely needed because these drugs are currently selected by trial and error, what is very inefficient and prejudicial for the patient and the healthcare system. Here, we have explored the discovery of protein biomarkers in serum from patients treated with infliximab, one of the major anti-TNF drugs. The study was based in a quantitative proteomics approach using 8-plex iTRAQ labeling. It combined depletion of the most abundant serum proteins, two-dimensional LC fractionation, protein identification and relative quantification with a hybrid Orbitrap mass spectrometer. This approach allowed the identification of 315 proteins of which 237 were confidently quantified with two or more peptides. The detection range covered up to 6 orders of magnitude including multiple proteins at the ng/mL level. A new set of putative biomarkers was identified comprising 14 proteins significantly more abundant in the non-responder patients. The differential proteins were enriched in apolipoproteins, components of the complement system and acute phase reactants. These results show the feasibility of this approach and provide a set of candidates for validation as biomarkers for the classification of RA patients before the beginning of treatment, so that anticipated non-responders could be treated with an alternative drug.

Tiger prawn, Penaeus monodon, and banana white prawn, Fenneropenaeus indicus—two of the most important commercial shrimp species—share world distribution, habitat, and similar morphometric characters. Because of commercial regulations about labeling and traceability requirements, species authentication methods are of great interest to fisheries and seafood industries. In this study, peptide mass fingerprints of both species are compared, using Matrix Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS) after trypsin digestion of a protein resolved by two-dimensional gel electrophoresis. Though low variability among these species is observed, two peptides of mass 1077.6 and 1205.7 Da are found to be useful to differentiate them.

Response to treatment of rheumatoid arthritis shows large inter-individual variability. This heterogeneity is observed with all the anti-rheumatic drugs, including the commonly used TNF inhibitors. It seems that drug-specific and target-specific factors lead individual patients to respond or not to a given drug, although this point has been challenged. The search of biomarkers distinguishing responders from non-responders has included shotgun proteomics of serum, as a previous study of response to infliximab, an anti-TNF antibody. Here, we have used the same study design and technology to search biomarkers of response to a different anti-TNF antibody, adalimumab, and we have compared the results obtained for the two anti-TNF drugs. Search of biomarkers of response to adalimumab included depletion of the most abundant serum proteins, 8-plex isobaric tag for relative and absolute quantitation (iTRAQ) labeling, two-dimensional liquid chromatography fractionation and relative quantification with a hybrid Orbitrap mass spectrometer. With this approach, 264 proteins were identified in all the samples with at least 2 peptides and 95% confidence. Nine proteins showed differences between non-responders and responders (P < 0.05), representing putative biomarkers of response to adalimumab. These results were compared with the previous study of infliximab. Surprisingly, the non-responder/responder differences in the two studies were not correlated (rs = 0.07; P = 0.40). This overall independence with all the proteins showed two identifiable components. On one side, the putative biomarkers of response to either adalimumab or infliximab, which were not shared and showed an inverse correlation (rs = -0.69; P = 0.0023). On the other, eight proteins showing significant non-responder/responder differences in the analysis combining data of response to the two drugs. These results identify new putative biomarkers of response to treatment of rheumatoid arthritis and indicate that they are notably drug-specific. ISSN:1932-6203

Lung cancer currently ranks as the neoplasia with the highest global mortality rate. Although some improvements have been introduced in recent years, new advances in diagnosis are required in order to increase survival rates. New mildly invasive endoscopy-based diagnostic techniques include the collection of bronchoalveolar lavage fluid (BALF), which is discarded after using a portion of the fluid for standard pathological procedures. BALF proteomic analysis can contribute to clinical practice with more sensitive biomarkers, and can complement cytohistological studies by aiding in the diagnosis, prognosis, and subtyping of lung cancer, as well as the monitoring of treatment response. The range of quantitative proteomics methodologies used for biomarker discovery is currently being broadened with the introduction of data-independent acquisition (DIA) analysis-related approaches that address the massive quantitation of the components of a proteome. Here we report for the first time a DIA-based quantitative proteomics study using BALF as the source for the discovery of potential lung cancer biomarkers. The results have been encouraging in terms of the number of identified and quantified proteins. A panel of candidate protein biomarkers for adenocarcinoma in BALF is reported; this points to the activation of the complement network as being strongly over-represented and suggests this pathway as a potential target for lung cancer research. In addition, the results reported for haptoglobin, complement C4-A, and glutathione S-transferase pi are consistent with previous studies, which indicates that these proteins deserve further consideration as potential lung cancer biomarkers in BALF. Our study demonstrates that the analysis of BALF proteins by liquid chromatography–tandem mass spectrometry (LC–MS/MS), combining a simple sample pre-treatment and SWATH DIA MS, is a useful method for the discovery of potential lung cancer biomarkers. Significance Bronchoalveolar lavage fluid (BALF) analysis can contribute to clinical practice with more sensitive biomarkers, thus complementing cytohistological studies in order to aid in the diagnosis, prognosis, and subtyping of lung cancer, as well as the monitoring of treatment response. Here we report a panel of candidate protein biomarkers for adenocarcinoma in BALF. Forty-four proteins showed a fold-change higher than 3.75 among adenocarcinoma patients compared with controls. This report is the first DIA-based quantitative proteomics study to use bronchoalveolar lavage fluid (BALF) as a matrix for discovering potential biomarkers. The results are encouraging in terms of the number of identified and quantified proteins, demonstrating that the analysis of BALF proteins by a SWATH approach is a useful method for the discovery of potential biomarkers of pulmonary diseases.

32 pages, 3 tables, 1 figure, Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. The present review addresses the recent developments regarding the application of DNA- and protein-based methods for the detection of allergenic ingredients in foods. The fitness-for-purpose of reviewed methodology will be discussed, and future trends will be highlighted. Special attention will be given to the evaluation of the potential of newly developed and promising technologies that can improve the detection and identification of allergenic ingredients in foods, such as the use of biosensors and/or nanomaterials to improve detection limits, specificity, ease of use, or to reduce the time of analysis. Such rapid food allergen test methods are required to facilitate the reliable detection of allergenic ingredients by control laboratories, to give the food industry the means to easily determine whether its product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred.

17 pages, 5 figures, 3 tables, Authentication of food products has demanded great attention by consumers and the food industry in recent years. Food components may be adulterated, leading to mislabeling and commercial fraud, especially in fish and shellfish products, where the substitution of high quality species by lower quality ones is frequent. Many regulations have appeared in different countries to fight against this issue. Although many analytical techniques have been applied to food authentication, new molecular approaches are emerging currently. In this sense, foodomics, defined as a new discipline that studies the food and nutrition domains through the application of advanced omics technologies in order to improve consumer's well-being, health, and confidence, can help in the authentication of food products. This chapter is a comprehensive overview of the applications of foodomics-related technologies, namely genomics, metabolomics, and proteomics, to the authentication of seafood products

9 páginas, 4 tablas, 2 figuras, Three factors defining the traceability of a food product are production method (wild or farmed), geographical origin and biological species, which have to be checked and guaranteed, not only in order to avoid mislabelling and commercial fraud, but also to address food safety issues and to comply with legal regulations. The aim of this study was to determine whether these three factors could be differentiated in shrimps using stable isotope ratio analysis of carbon and nitrogen and/or multi-element composition. Different multivariate statistics methods were applied to different data subsets in order to evaluate their performance in terms of classification or predictive ability. Although the success rates varied depending on the dataset used, the combination of both techniques allowed the correct classification of 100% of the samples according to their actual origin and method of production, and 93.5% according to biological species. Even though further studies including a larger number of samples in each group are needed in order to validate these findings, we can conclude that these methodologies should be considered for studies regarding seafood product authenticity

40 pages, 2 figures, 2 tables, The close relationship between nutrition and health has led to changes in the habits of consumers, who are demanding products that meet their dietary and nutritional preferences. In light of this demand, both academia and food industries face a new challenge: the need for developing strategies and products that are not only safe, but also contribute to the maintenance of good health and that can even prevent the development of specific diseases. Advances in proteome analysis offer multiple applications that can be used to meet the challenge. These applications can be divided into three main topics: (i) quality and food safety control; (ii) the study of changes in food components as a consequence of food processing; and (iii) nutritional aspects and characterization of new food ingredients with beneficial effects on human health. In this chapter, we illustrate how proteomic tools can be applied to these three main food science topics

12 páginas, 4 figuras, 2 tablas, Genomic and proteomic techniques for species identification of meat and seafood products are being widely used. In this study, a genomic approach was used to differentiate Pandalus borealis (the Northern shrimp), which belongs to the superfamily Pandaloidea, from 30 crustaceans consisting of 19 commercially relevant prawns/shrimps species that belong to the superfamily Penaeoidea, which include the families Penaeidae and Solenoceridae, and 11 other crustacean species, including prawns, shrimps, lobsters, and crabs. For this purpose, a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) method was designed based on the amplification of the 16S rRNA/tRNAVal/12S rRNA mitochondrial regions using the primers 16S-CruF and 16S-CruR. The 966-bp PCR products were produced and cleaved with the restriction enzymes AluI, TaqI, and HinfI, which provided species-specific restriction patterns. In addition, a proteomic approach, based on matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) and electrospray ionization–ion trap (ESI–IT) mass spectrometry, was used to identify and characterize new P. borealis-specific peptides that could be useful as potential markers of this species in protein-based detection methods. To our knowledge, this is the first time a molecular method has been successfully applied to identify a wide range of prawn and shrimp species, including P. borealis, for either whole individuals or processed products. However, validation of the methods proposed here is required by applying them to a larger sample of individuals from different populations and geographic origins in order to avoid mainly false-negative results, National Food Program of the INIA (Spanish Ministry for Education, project CAL-03-030-C2) and the PGIDIT Research Program in Marine Resources (project PGIDIT04RMA261004PR) of the Xunta de Galicia (Galician Council for Industry Commerce and Innovation)

5 páginas, 2 figuras, 2 tablas, Polymerase chain reaction-based methodologies have been developed for the identification of three commercially- relevant penaeid shrimp species, these were: Litopenaeus vannamei, Penaeus monodon and Fenneropenaeus indicus in food products. Such three species represent more than 80% of the whole farmed shrimp production worldwide and may be fraudulently replaced by species exhibiting lower value such as Litopenaeus stylirostris, Penaeus semisulcatus and Fenneropenaeus merguiensis, respectively. For it, preliminary sequencing of a mitochondrial sequence of ca. 530 bp in the 16S rRNA/tRNAVal mitochondrial region was performed in nearly 20 penaeid shrimp species of commercial relevance. Careful analysis of such sequences allowed the design of primers PNVF/PNVR, which allowed the combined identification of P. monodon and L. vannamei, and PNIF/PNIR, which allowed the specific identification of F. indicus. In addition, P. monodon and L. vannamei could be easily differentiated by either restriction with TspE1 or by amplification with novel primers MPNF/MPNR, specific for P. monodon. The proposed specific methods improve current general identification methods of these species based on more general RFLP analyses. In addition, these methods can be easily completed in less than 8 h., The authors thank the financial support from the National Food Program of the INIA (Spanish Ministry for Education) (Project CAL-03-030-C2-1) and from the PGIDIT Research Program in Marine Resources (Project PGIDIT04RMA261004PR) of the Galician Government (Xunta de Galicia-Galician Council for Industry, Commerce and Innovation).

Selected tandem mass spectrometry (MS/MS) ion monitoring (SMIM) is the most suitable scanning mode to detect known peptides in complex samples when an ion-trap mass spectrometer is the instrument used for the analysis. In this mode, the MS detector is programmed to perform continuous MS/MS scans on one or more selected precursors, either during a selected time interval, or along the whole chromatographic run. MS/MS spectra are recorded, so virtual multiple reaction monitoring chromatogram traces for the different fragment ions can be plotted. In this work, a shotgun proteomics approach was applied to the detection of previously characterized species-specific peptides from different seafood species. The proposed methodology makes use of high intensity focused ultrasound-assisted trypsin digestion for ultra fast sample preparation, peptide separation and identification by reverse phase capillary LC coupled to an ion-trap working in the SMIM scanning mode. This methodology was applied to the differential classification of seven commercial, closely related, species of Decapoda shrimps proving to be an excellent tool for seafood product authentication, which may be used by fisheries and manufacturers to provide a fast and effective identification of the specimens, guaranteeing the quality and safety of foodstuffs to consumers.

10 pages, 5 figures, 3 tables, The effect of a previous hydrostatic high pressure (HHP) treatment on hydrolysis, breakdown and aggregation events in chemical constituents of chilled farmed coho salmon (Oncorhynchus kisutch) was studied. Three different HHP conditions were applied (135 MPa-30 s; 170 MPa-30 s; 200 MPa-30 s; treatments T-1, T-2 and T-3, respectively) and compared to untreated fish for a 20 day-chilled storage. Nucleotide degradation was important during the chilled storage in all kinds of samples; however, the K value did not afford differences related to previous pressure applied. HHP treatment led to an increased free fatty acid (FFA) formation (day 0 values); contrary, an inhibitory effect on FFA formation could be observed at the end of the storage (15-20 days) in T-3 treated fish as a result of microbial activity inhibition. A marked decrease in sarcoplasmic protein content was evident in samples corresponding to T-2 and T-3 treatments; the SDS-PAGE analysis of such protein fraction showed a partial loss of a band corresponding to 29 kDa. This band was excised, digested with trypsin, analysed by tandem mass spectrometry and identified as phosphoglycerate mutase, This work was supported by the Universidad de Chile (Chile)-Consejo Superior de Investigaciones Cientı´ficas (Spain) program (Project 2006 CL 0034) and by the FONDECYT program (Chile; project number: 1080626).

The identification of commercial shrimp species is a relevant issue to ensure correct labeling, maintain consumer confidence and enhance the knowledge of the captured species, benefiting both, fisheries and manufacturers. A proteomic approach, based on 2DE, tryptic in-gel digestion, MALDI-TOF MS, and ESI-MS/MS analyses, is proposed for the identification of shrimp species with commercial interest. MALDI-TOF peptide mass fingerprint from arginine kinase tryptic digests were used for the identification of seven commercial, closely related species of Decapoda shrimps. Further identification and characterization of these peptides was performed by CID on an ESI-IT instrument, database search and de novo sequence interpretation, paying special attention to differential, species-specific peptides. Fisheries and manufacturers may take advantage of this methodology as a tool for a rapid and effective seafood product identification and authentication, providing and guaranteeing the quality and safety of the foodstuffs to consumers.

8 páginas, 4 figuras, 2 tablas, A proteomic approach aimed at species identification and taxonomic analysis of shrimp species of commercial interest is presented. Six different species belonging to the order Decapoda were considered. Preliminary, two-dimensional gel electrophoresis (2-DE) analysis of the sarcoplasmic proteome revealed interspecific variability in the isoelectric point (pI) of arginine kinase. For this reason, arginine kinase spot was selected as a potential molecular marker and subjected to tryptic digestion followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) peptide mass fingerprinting (PMF) analysis. Arginine kinase PMF allowed the differentiation of the six species studied. Four samples of commercial origin obtained in local markets were analyzed to validate the methodology. The PMF cluster analysis also provided information about the phylogenetic relationships in these species. The application of this methodology may be of interest for the differentiation and taxonomic analysis of shrimp species complementing DNA-based phylogenetic studies., This work was supported by the National Food Program of the INIA (Spanish Ministry for Education and Science) (Project CAL-03-030-C2-2) and by the PGIDIT Research Program in Marine Resources (Project PGIDIT04RMA261004PR) of the Xunta de Galicia (Galician Council for Industry, Commerce and Innovation). I.O. is supported by the FPU program (AP-2004-5826) and B.C. is supported by the RyC program, under the auspices of the Spanish MICINN

Background Biological therapies have been a great advance in Rheumatoid Arthritis (RA) treatment. However, up to a third of the RA patients do not respond to a given biologic and a method to predict response is not yet available. Therefore, the only approach to select effective biological drugs is by trial and error. In addition, assessment of response requires months of treatment making this approach very inefficient and detrimental for patients and healthcare systems. Thus, biomarkers for prediction of anti-TNF response at an early stage are sorely needed. A potential source for these biomarkers is the analysis of serum proteins as shown by the report of six plasma proteins (only two of them were identified) detected with the SELDI-TOF technology1, which are potential biomarkers of response to anti-TNF therapy in RA patients. Objectives To search for biomarkers of infliximab response by means of a very precise and sensitive quantitative proteomics technology applied to serum samples of patients with RA taken before the beginning of treatment. Methods Donors with RA according to ACR criteria that never had received biologics were enrolled in the study. Serum was collected and stored at -80°C before the beginning of treatment. Donors were treated with infliximab and clinical response, assessed by the EULAR criteria, was determined six months later. Eight samples showing extreme responses were selected: 4 with good clinical response and 4 with no response. Each of the serum samples was depleted of the six most abundant serum proteins and, after tryptic digestion, they were differentially labeled with the iTRAQ (Isobaric Tags for Relative and Absolute Quantification) isotopic labels. The peptide mixture was fractionated by means of 2D-LC (strong cation exchange chromatography and reversed phase chromatography), on-line coupled with an Orbitrap Velos mass spectrometer. ProteinPilot 4.0 software was used for the identification and relative quantification of proteins. Results Obtained mass spectra were compared with the human protein database, enabling the identification of 315 different proteins that were present in all the samples. Most of these proteins were of medium and low abundance in serum. A total of 289 proteins could be quantified in all samples using the iTRAQ differential labeling and 17 of them showed concentration differences between responders and no-responders (p

Background Epigenetic regulation influences gene expression and can affect disease course and treatment response. Global DNA methylation of T cells from patients with RA is decreased 1 . This hypomethylation could favour inflammation and tissue destruction through aberrant gene expression. However, little is known about this process and whether other blood cells are at play. Methotrexate (MTX) is widely used for the treatment of RA but its mechanism of action is not completely understood. It could include increased DNA methylation as already suggested in unseparated blood cells 2 . Objectives To evaluate global DNA methylation and expression of enzymes involved in DNA methylation (DNMTs) and demethylation (GADD45a) in five peripheral blood cell subpopulations from patients with early RA before and after MTX treatment. Methods T and B lymphocytes, monocytes, NK cells and granulocytes were separated by magnetic isolation from blood of 20 patients with early RA (baseline and after 4-6 weeks of MTX treatment) and 17 healthy controls (matched for gender and age). Genomic DNA and RNA were simultaneously extracted. Global methylation levels were assessed by HPLC-ESI-MRM and gene expression of DNMT1 , DNMT3A , DNMT3B and GADD45A was determined by real time PCR. Results Global DNA methylation of T cells in RA patients was significantly lower than in healthy controls (3.97 % ± 0.35 versus 4.23 % ± 0.45, P = 0.023). This difference was associated with about half DNMT1 expression in patients than in controls ( P = 0.014). DNMT1 expression was also lower in monocytes ( P = 0.0005) and B cells ( P = 0.013) from RA patients, although global DNA methylation was not significantly different. After 4-6 weeks of MTX treatment, DNA methylation in T cells of RA patients was increased to levels comparable with controls (4.23 % ± 0.43, P = 0.007 compared with baseline). DNA methylation also increased in monocytes ( P = 0.023). DNMT1 expression levels were also increased in T cells after MTX treatment ( P = 0.027). Conclusions Compared with healthy controls, T cells of early RA patients show less DNA methylation associated with lower expression of the main methylation maintenance enzyme DNMT1 . These changes regress after MTX treatment. Similar but less prominent changes are observed in B cells and monocytes. These differences in peripheral blood cells could reflect important mechanisms both in disease evolution and its control by MTX. References Richardson B et al. Arthritis Rheum. 1990;33:1665-73 Kim YI et al. J Lab Clin Med. 1996;128:165-72 Acknowledgements Funding was provided by grant PI11/01048 and PI12/01909 of the Instituto de Salud Carlos III (Spain) that are partially financed by the European Regional Development Fund of the European Union. Disclosure of Interest None Declared