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1.
Int J Mol Sci ; 24(10)2023 May 12.
Article En | MEDLINE | ID: mdl-37240037

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.


Antineoplastic Agents , Crohn Disease , Humans , Crohn Disease/drug therapy , Tumor Necrosis Factor Inhibitors/therapeutic use , Vinculin , Tumor Necrosis Factor-alpha/therapeutic use , Antineoplastic Agents/therapeutic use , Remission Induction , Infliximab/therapeutic use
2.
Nutrients ; 15(4)2023 Feb 08.
Article En | MEDLINE | ID: mdl-36839217

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.


Inulin , Transcriptome , Animals , Mice , Inulin/pharmacology , Proteomics , Macrophages/metabolism , Inflammation/metabolism , Anti-Inflammatory Agents/pharmacology , Prebiotics
3.
Int J Biol Macromol ; 225: 1280-1290, 2023 Jan 15.
Article En | MEDLINE | ID: mdl-36427620

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 values <0.05 and logFC >1). In conclusion, we have been able to describe several behaviors of the whole HDL proteome during the postprandial hyperlipidemic metabolism.


Hyperlipidemias , Lipoproteins, HDL , Humans , Male , Proteome , Healthy Volunteers , Chromatography, Liquid , Proteomics , Tandem Mass Spectrometry , Postprandial Period , Triglycerides
4.
Int J Mol Sci ; 23(15)2022 Aug 05.
Article En | MEDLINE | ID: mdl-35955870

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.


Lung Neoplasms , Proteomics , Biomarkers, Tumor/metabolism , Humans , Lung Neoplasms/diagnosis , Lung Neoplasms/metabolism , Proteome/metabolism , Proteomics/methods , Sputum/chemistry
5.
Methods Mol Biol ; 2259: 205-213, 2021.
Article En | MEDLINE | ID: mdl-33687717

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.


Bacteria/isolation & purification , Bacterial Proteins/analysis , Food Microbiology , Proteomics/methods , Bacterial Proteins/isolation & purification , Chromatography, Liquid/methods , Software , Spectrometry, Mass, Electrospray Ionization/methods , Tandem Mass Spectrometry/methods
6.
Toxicol Res (Camb) ; 9(4): 474-483, 2020 Jul.
Article En | MEDLINE | ID: mdl-32905142

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.

7.
Aging (Albany NY) ; 12(12): 11277-11286, 2020 06 23.
Article En | MEDLINE | ID: mdl-32575076

Coronavirus disease 2019 (COVID-19), caused by an outbreak of the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in Wuhan, China, has led to an unprecedented health and economic crisis worldwide. To develop treatments that can stop or lessen the symptoms and severity of SARS-CoV-2 infection, it is critical to understand how the virus behaves inside human cells, and so far studies in this area remain scarce. A recent study investigated translatome and proteome host cell changes induced in vitro by SARS-CoV-2. Here, we use the publicly available proteomics data from this study to re-analyze the in vitro cellular consequences of SARS-CoV-2 infection by impact pathways analysis and network analysis. Notably, proteins linked to the inflammatory response, but also proteins related to chromosome segregation during mitosis, were found to be altered in response to viral infection. Upregulation of inflammatory response proteins is in line with the propagation of inflammatory reaction and lung injury that is observed in advanced stages of COVID-19 patients and which worsens with age.


Betacoronavirus/physiology , Coronavirus Infections/virology , Pneumonia, Viral/virology , Proteomics , Aging , Angiotensin-Converting Enzyme 2 , COVID-19 , Gene Expression Regulation , Gene Regulatory Networks , Humans , Inflammation , Pandemics , Peptidyl-Dipeptidase A/metabolism , SARS-CoV-2 , Virus Replication
8.
Nutrients ; 11(12)2019 Dec 06.
Article En | MEDLINE | ID: mdl-31817645

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) - 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.


Antineoplastic Agents/pharmacology , Arachidonic Acid/pharmacology , Docosahexaenoic Acids/pharmacology , Monoglycerides/pharmacology , Cell Death/drug effects , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , DNA Replication/drug effects , Drug Screening Assays, Antitumor , HT29 Cells , Humans , Mass Spectrometry , Proteasome Endopeptidase Complex/drug effects , Proteomics
9.
J Cell Mol Med ; 23(12): 8219-8232, 2019 12.
Article En | MEDLINE | ID: mdl-31560832

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.


Biomarkers, Tumor/metabolism , Colorectal Neoplasms/metabolism , Proteome/metabolism , Proteomics/methods , Aged , Biomarkers, Tumor/genetics , Colorectal Neoplasms/classification , Colorectal Neoplasms/genetics , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Ontology , Gene Regulatory Networks , Humans , Immune Evasion , Kaplan-Meier Estimate , Male , Middle Aged , Neoplasm Metastasis , Prognosis , Proteome/genetics
10.
J Agric Food Chem ; 67(14): 3854-3864, 2019 Apr 10.
Article En | MEDLINE | ID: mdl-30901215

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.


DNA Barcoding, Taxonomic/methods , DNA/genetics , Food Contamination/analysis , Plants/genetics , Animals , Food , Meat/analysis , Plants/chemistry , Plants/classification
11.
J Proteomics ; 193: 217-229, 2019 02 20.
Article En | MEDLINE | ID: mdl-30626528

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.


Biomarkers, Tumor/metabolism , G2 Phase Cell Cycle Checkpoints/drug effects , Lactones/pharmacology , M Phase Cell Cycle Checkpoints/drug effects , Microtubule-Associated Proteins/metabolism , Neoplasm Proteins/metabolism , Prostatic Neoplasms, Castration-Resistant , Humans , Male , PC-3 Cells , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/metabolism , Prostatic Neoplasms, Castration-Resistant/pathology , Proteomics
12.
Hum Mol Genet ; 27(17): 3046-3059, 2018 09 01.
Article En | MEDLINE | ID: mdl-29878202

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 and 5-methylcytosine (5mC) in human primary samples from 12 non-tumoral brains and 53 gliomas. We found that the levels of 5hmC identified in non-tumoral samples were significantly reduced in gliomas. Strikingly, hypo-hydroxymethylation at 4627 (9.3%) CpG sites was associated with aberrant DNA hypermethylation and was strongly enriched in CpG island shores. 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. As this 5mC gain is inversely correlated with loss of 5hmC and has not been identified with classical sodium bisulfite-based technologies, we conclude that our data identifies a novel 5hmC-dependent type of aberrant DNA hypermethylation in glioma.


5-Methylcytosine/analogs & derivatives , Biomarkers, Tumor/genetics , DNA Methylation , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , Genome, Human , Glioma/pathology , 5-Methylcytosine/metabolism , Case-Control Studies , CpG Islands , Glioma/genetics , Glioma/metabolism , Humans
13.
J Agric Food Chem ; 66(24): 6003-6012, 2018 Jun 20.
Article En | MEDLINE | ID: mdl-29804451

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.


Antineoplastic Agents/pharmacology , Arachidonic Acids/pharmacology , Colorectal Neoplasms/genetics , Docosahexaenoic Acids/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/physiopathology , Humans , Proteomics
14.
J Proteomics ; 175: 105-113, 2018 03 20.
Article En | MEDLINE | ID: mdl-29317355

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. SIGNIFICANCE: 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 peak <7.3ng/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.


Apolipoproteins/analysis , Human Growth Hormone/deficiency , Machine Learning , Mass Spectrometry/methods , Adolescent , Age of Onset , Algorithms , Apolipoproteins A/analysis , Biomarkers/blood , Case-Control Studies , Child , Child, Preschool , Female , Humans , Male , beta-Thromboglobulin/analysis
15.
J Proteomics ; 152: 138-149, 2017 01 30.
Article En | MEDLINE | ID: mdl-27989941

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.


Biomedical Research/methods , Chromatography, Liquid/methods , Proteomics/methods , Biomedical Research/standards , Chromatography, Liquid/standards , Observer Variation , Proteomics/organization & administration , Proteomics/standards , Reference Standards , Reproducibility of Results , Research/standards
16.
J Proteomics ; 147: 212-225, 2016 09 16.
Article En | MEDLINE | ID: mdl-27389853

UNLABELLED: 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.


Food Analysis/methods , Food Handling/methods , Proteomics/methods , Animals , Biomarkers/analysis , Food Handling/standards , Food Technology , Humans
17.
PLoS One ; 11(4): e0153140, 2016.
Article En | MEDLINE | ID: mdl-27050469

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.


Adalimumab/therapeutic use , Antirheumatic Agents/therapeutic use , Arthritis, Rheumatoid/drug therapy , Biomarkers/blood , Blood Proteins/metabolism , Infliximab/therapeutic use , Adult , Aged , Arthritis, Rheumatoid/blood , Female , Humans , Male , Middle Aged
18.
Arthritis Res Ther ; 17: 233, 2015 Aug 29.
Article En | MEDLINE | ID: mdl-26330155

INTRODUCTION: DNA methylation is an epigenetic mechanism regulating gene expression that has been insufficiently studied in the blood of rheumatoid arthritis (RA) patients, as only T cells and total peripheral blood mononuclear cells (PBMCs) from patients with established RA have been studied and with conflicting results. METHOD: Five major blood cell subpopulations: T, B and NK cells, monocytes, and polymorphonuclear leukocytes, were isolated from 19 early RA patients and 17 healthy controls. Patient samples were taken before and 1 month after the start of treatment with methotrexate (MTX). Analysis included DNA methylation with high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (HPLC-ESI-MS/MS-SRM) and expression levels of seven methylation-specific enzymes by quantitative polymerase chain reaction (qPCR). RESULTS: Disease-modifying anti-rheumatic drug (DMARD)-naïve early RA patients showed global DNA hypomethylation in T cells and monocytes, together with a lower expression of DNA methyltrasnferase 1 (DNMT1), the maintenance DNA methyltransferase, which was also decreased in B cells. Furthermore, significantly increased expression of ten-eleven translocation1 (TET1), TET2 and TET3, enzymes involved in demethylation, was found in monocytes and of TET2 in T cells. There was also modest decreased expression of DNMT3A in B cells and of growth arrest and DNA-damage-inducible protein 45A (GADD45A) in T and B cells. Treatment with MTX reverted hypomethylation in T cells and monocytes, which were no longer different from controls, and increased global methylation in B cells. In addition, DNMT1 and DNMT3A showed a trend to reversion of their decreased expression. CONCLUSIONS: Our results confirm global DNA hypomethylation in patients with RA with specificity for some blood cell subpopulations and their reversal with methotrexate treatment. These changes are accompanied by parallel changes in the levels of enzymes involved in methylation, suggesting the possibility of regulation at this level.


Arthritis, Rheumatoid/drug therapy , B-Lymphocytes/drug effects , DNA Methylation/drug effects , Leukocytes, Mononuclear/drug effects , Methotrexate/therapeutic use , T-Lymphocytes/drug effects , Adult , Aged , Antirheumatic Agents/therapeutic use , Arthritis, Rheumatoid/blood , Arthritis, Rheumatoid/genetics , B-Lymphocytes/metabolism , Cell Cycle Proteins/genetics , Chromatography, High Pressure Liquid , DNA (Cytosine-5-)-Methyltransferase 1 , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methyltransferase 3A , DNA-Binding Proteins/genetics , Dioxygenases/genetics , Female , Gene Expression Regulation/drug effects , Humans , Leukocytes, Mononuclear/metabolism , Male , Middle Aged , Mixed Function Oxygenases , Nuclear Proteins/genetics , Proto-Oncogene Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Spectrometry, Mass, Electrospray Ionization , T-Lymphocytes/metabolism , Time Factors , DNA Methyltransferase 3B
19.
Food Chem ; 170: 145-53, 2015 Mar 01.
Article En | MEDLINE | ID: mdl-25306329

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.


Artemia/chemistry , Isotopes/analysis , Seafood/analysis , Animals , Geography
20.
J Proteomics ; 77: 372-82, 2012 Dec 21.
Article En | MEDLINE | ID: mdl-23000593

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.


Antibodies, Monoclonal/administration & dosage , Antirheumatic Agents/administration & dosage , Arthritis, Rheumatoid/blood , Arthritis, Rheumatoid/drug therapy , Blood Proteins/metabolism , Proteome/metabolism , Biomarkers/blood , Female , Humans , Infliximab , Male , Proteomics/methods
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