Proteomics: translating genes into cellular functions to understand biology and disease

The sequencing of the human genome has provided a privileged envision of our own biology. However, much remains to be explored to fully define a complete map of cell functions, their regulatory mechanisms and the alterations participating in disease. As proteins are primary effectors of cellular pathways, the study of the proteome based on a systems biology strategy has opened new perspectives in biomedical research. Proteome complexity, the wide dynamic range of its components as well as their inherent physicochemical nature precludes the coverage of the entire protein set of a living organism on a single experiment. However, the astonishing progress in protein/peptide fractionation combined with mass spectrometry in the past decade allow unprecedented proteome coverage and measurement accuracy. Proteomics emerges, therefore, as an exciting tool in biomedical research. In this seminar basic concepts and state of the art methods in proteomics will be revised as well as their application to the analysis of complex cell and tissue proteomes. One carbon metabolism (1CM) was prioritized as a central pathway associated to the progression of liver disorders. Impairment of the methylation capacity of liver cells lead to the identification of 216 differential proteins that suggest deregulation of cellular pathways as those mediated by ERK or NFκB. R-methyl proteome analysis lead to the identification of 74 differentially methylated proteins, including 116 new methylation sites. Inhibition of RNA binding proteins methylation is especially relevant. Moreover, quantification of 1CM enzymes by SRM in the liver revealed a tissue specific expression profile as well as a significant remodelling in mice upon CCl4 induced liver injury and in liver tumours. It is then tempting to suggest that impairment of 1CM is a bad prognostic hallmark in cancer. The systematic monitorization of one carbon metabolism in the liver may probe its usefulness for the assessment of liver parenchymal cells homeostasis.Fil: Paradela, Alberto. Consejo Superior de Investigaciones Científicas; EspañaFil: Ambao, Veronica Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Granero, Ignacio. Consejo Superior de Investigaciones Científicas; EspañaFil: Guerrero, Laura. Consejo Superior de Investigaciones Científicas; EspañaFil: Corrales, Fernando J.. Consejo Superior de Investigaciones Científicas; España7th WorkshopMadridEspañaConsejo Superior de Investigaciones CientíficasCentre National de la Recherche Scientifiqu

Miape web toolkit: a user-friendly work frame to conncect and share proteomics standards in the lab

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Plasma Proteomics: optimized experimental design in cardiovascular disease

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Isotope-coded protein labelling (ICPL) as a tool for the quantitative analysis of a bacterial proteome

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Analyzing materials in the microscopes: From the Sorby thin sections up to the nondestructive large chambers

Proceedings of The Fifth Saudi International Meeting on Frontiers of Physics (SIMFP2016)One hundred and sixty five years ago, Henry Clifton Sorby developed a revolutionary technique to prepare thin and polished sections of rocks and minerals to be observed by transmission and reflectance in the optical polarized light microscopes. Nowadays, Sorby¿s methods are still employed for near all inorganic materials with scarce modifications. The optical microscopy subject is essential for rocks analysis; it is an irreplaceable technique for specific complex samples, such as twinned-exsolved feldspars and it must be preserved in the geosciences curriculum. However, for many valuable and common specimens we observe a strong growing of non-destructive techniques coupled to the large chambers of modern microscopes including new chemical, molecular, luminescent, metrical, imaging and structural probes. Suitable explanations for the thin sections decreasing in research laboratories could be: (i) the undesirable cutting of the specimen; (ii) the contamination of surfaces by abrasives and glues; (iii) the etching with dangerous acids; (iv) the slow and painful works of cutting, grinding and polishing specimens; (v) the necessary protection of valuable specimens, (vi) the need for immediate analytical results.Peer Reviewe

A systematic approach to evaluate metabolic conversions of amino acids in silac experiments

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Monitoring one-carbon metabolism by mass spectrometry to assess liver function and disease

Precision medicine promises to overcome the constraints of the traditional “one-for-all” healthcare approach through a clear understanding of the molecular features of a disease, allowing for innovative and tailored treatments. State-of-the-art proteomics has the potential to accurately explore the human proteome to identify, quantify, and characterize proteins associated with disease progression. There is a pressing need for informative biomarkers to diagnose liver disease early in its course to prevent severe disease for which no efficient treatment is yet available. Here, we propose the concept of a cellular pathway as a functional biomarker, whose monitorization may inform normal and pathological status. We have developed a standardized targeted selected-reaction monitoring assay to detect and quantify 13 enzymes of one-carbon metabolism (1CM). The assay is compliant with Clinical Proteomics Tumor Analysis Consortium (CPTAC) guidelines and has been included in the protein quantification assays that can be accessed through the assay portal at the CPTAC web page. To test the feasibility of the assay, we conducted a retrospective, proof-of-concept study on a collection of liver samples from healthy controls and from patients with cirrhosis or hepatocellular carcinoma (HCC). Our results indicate a significant reconfiguration of 1CM upon HCC development resulting from a process that can already be identified in cirrhosis. Our findings indicate that the systematic and integrated quantification of 1CM enzymes is a promising cell function-based biomarker for patient stratification, although further experiments with larger cohorts are needed to confirm these findings.Fil: Guerrero, Laura. Consejo Superior de Investigaciones Científicas; EspañaFil: Sangro, Bruno. Universidad de Navarra; EspañaFil: Ambao, Veronica Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Granero, José Ignacio. Consejo Superior de Investigaciones Científicas; EspañaFil: Ramos Fernández, Antonio. No especifíca;Fil: Paradela, Alberto. Consejo Superior de Investigaciones Científicas; EspañaFil: Corrales, Fernando J.. Consejo Superior de Investigaciones Científicas; Españ

iTRAQ Quantitative Proteomic Analysis of Vitreous from Patients with Retinal Detachment

Rhegmatogenous retinal detachment (RRD) is a potentially blinding condition characterized by a physical separation between neurosensory retina and retinal pigment epithelium. Quantitative proteomics can help to understand the changes that occur at the cellular level during RRD, providing additional information about the molecular mechanisms underlying its pathogenesis. In the present study, iTRAQ labeling was combined with two-dimensional LC-ESI-MS/MS to find expression changes in the proteome of vitreous from patients with RRD when compared to control samples. A total of 150 proteins were found differentially expressed in the vitreous of patients with RRD, including 96 overexpressed and 54 underexpressed. Several overexpressed proteins, several such as glycolytic enzymes (fructose-bisphosphate aldolase A, gamma-enolase, and phosphoglycerate kinase 1), glucose transporters (GLUT-1), growth factors (metalloproteinase inhibitor 1), and serine protease inhibitors (plasminogen activator inhibitor 1) are regulated by HIF-1, which suggests that HIF-1 signaling pathway can be triggered in response to RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Nevertheless, the differentially expressed proteins found in this study suggest that different mechanisms are activated after RRD to promote the survival of retinal cells through complex cellular responses.CENTRO-07-ST24-FEDER-002014; POCI-01-0145-FEDER-007491; CNB-CSIC proteomics lab is a member of ProteoRed, supported by PRB2-ISCIII grant [PT13/0001]; Novartis Farma-Produtos Farmacêuticos; PhD fellowship of Sciences Faculty financed by ICI and Santander.info:eu-repo/semantics/publishedVersio

Proteomics profiling of vitreous humor reveals complement and coagulation components, adhesion factors, and neurodegeneration markers as discriminatory biomarkers of vitreoretinal eye diseases

Funding Information: This project was supported by the University of Beira Interior— Health Sciences Research Centre (CICS-UBI) supported by FEDER funds through the POCI—COMPETE 2020—Operational Programme Competitiveness and Internationalisation in Axis I—Strengthening research, technological development, and innovation Project (POCI-01- 0145-FEDER-007491). CNB-CSIC proteomics lab is a member of Proteored, PRB2-ISCIII and is supported by grant PT13/0001, of the PE I +D+i 2013–2016, funded by ISCIII and FEDER. Publisher Copyright: Copyright © 2023 Santos, Ciordia, Mesquita, Cruz, Sousa, Passarinha, Tomaz and Paradela.Introduction: Diabetic retinopathy (DR) and age-related macular degeneration (AMD) are leading causes of visual impairment and blindness in people aged 50 years or older in middle-income and industrialized countries. Anti-VEGF therapies have improved the management of neovascular AMD (nAMD) and proliferative DR (PDR), no treatment options exist for the highly prevalent dry form of AMD.  Methods: To unravel the biological processes underlying these pathologies and to find new potential biomarkers, a label-free quantitative (LFQ) method was applied to analyze the vitreous proteome in PDR (n=4), AMD (n=4) compared to idiopathic epiretinal membranes (ERM) (n=4).  Results and discussion: Post-hoc tests revealed 96 proteins capable of differentiating among the different groups, whereas 118 proteins were found differentially regulated in PDR compared to ERM and 95 proteins in PDR compared to dry AMD. Pathway analysis indicates that mediators of complement, coagulation cascades and acute phase responses are enriched in PDR vitreous, whilst proteins highly correlated to the extracellular matrix (ECM) organization, platelet degranulation, lysosomal degradation, cell adhesion, and central nervous system development were found underexpressed. According to these results, 35 proteins were selected and monitored by MRM (multiple reaction monitoring) in a larger cohort of patients with ERM (n=21), DR/PDR (n=20), AMD (n=11), and retinal detachment (n=13). Of these, 26 proteins could differentiate between these vitreoretinal diseases. Based on Partial least squares discriminant and multivariate exploratory receiver operating characteristic (ROC) analyses, a panel of 15 discriminatory biomarkers was defined, which includes complement and coagulation components (complement C2 and prothrombin), acute-phase mediators (alpha-1-antichymotrypsin), adhesion molecules (e.g., myocilin, galectin-3-binding protein), ECM components (opticin), and neurodegeneration biomarkers (beta-amyloid, amyloid-like protein 2).publishersversionpublishe