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Jesús Vázquez and collaborators Jesús Vázquez Centro de Biología Molecular CBMSO. CSIC Protein Chemistry and Proteomics

Research Summary

Quantitative strategies relying on multidimensional chromatography coupled to mass spectrometry and 18O-stable isotope labeling are peptide-centric techniques that have emerged as a more robust and sensitive alternatives to the well-established gel-based techniques for the study of the dynamic proteome. Nevertheless, high-throughput peptide identification and quantification experiments require highly automated setups and data-processing algorithms. We are working in the implementation and development of these techniques and their application to the study of dynamic changes in the endothelial cell proteome during angiogenesis and to the analysis of posttranslational modifications induced in endothelium by nitrosative stress.

We have developed an integrated set of bioinformatics tools that includes a fully automated validation program (pRatio), which selects positive matches among tentative peptide identifications in databases from MS/MS data, and a quantification program (QuiXoT), which calculates expression ratios and introduces an innovative algorithm for computing labeling efficiency. In a representative experiment, we have identified and determined relative expression changes of more than 1.000 proteins (5.000 peptides) in HUVEC cells after 4 and 8h-estimulation with the angiogenic factor VEGF. Changes in relative expression levels were detected in proteins responding to external stimuli and involved in cell communication, including cell adhesion molecules. We have also developed methods for the large-scale study of differential expression of membrane proteins. We are also applying these technologies, in combination with peptide-centric affinity purification procedures, to study dynamic changes in proteomes at the level of posttranslational modifications. To date we have been able to characterize more than two hundred phosphorylation sites in the nuclear proteome of endothelial cells in the basal state. A similar approach has allowed the identification of several dozens of potentially nitrosylated proteins and the characterization of a tyrosine-nitration site using in vitro models of oxidative stress. Our results may contribute to the development of Second Generation Proteomics and could improve our knowledge of the molecular mechanisms underlying two important processes in the vascular endothelium, a key component of the cardiovascular system.

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