O potencial da rotulação metabólica de 15N para a pesquisa de esquizofrenia / The potential of 15N metabolic labeling for schizophrenia research

Pesquisas em psiquiatria ainda necessitam de estudos não dirigidos por hipóteses para revelar fundamentos neurobiológicos e biomarcadores moleculares para distúrbios psiquiátricos. Metodologias proteômicas disponibilizam uma série de ferramentas para esses fins. Apresentamos o princípio de rotulação metabólica utilizando 15N para proteômica quantitativa e suas aplicações em modelos animais de fenótipos psiquiátricos com um foco particular em esquizofrenia. Exploramos o potencial de rotulação metabólica por 15N em diferentes tipos de experimentos, bem como suas considerações metodológicas

Psychiatric research is in need of non-hypothesis driven approaches to unravel the neurobiological underpinnings and identify molecular biomarkers for psychiatric disorders. Proteomics methodologies constitute a state-of-the-art toolbox for biomarker discovery in psychiatric research. Here we present the principle of in vivo 15N metabolic labeling for quantitative proteomics experiments and applications of this method in animal models of psychiatric phenotypes, with a particular focus on schizophrenia. Additionally we explore the potential of 15N metabolic labeling in different experimental set-ups as well as methodological considerations of 15N metabolic labeling-based quantification studies

Identification of tumor markers with SILAC proteomics approaches / 基础医学与临床

Mass spectrometry-based quantitative proteomics has become an indispensable tool for tumor marker discovery.Stable isotope labeling with amino acid in cell culture(SILAC),as a representative of in vivo metabolic labeling strategy,is a simple and relatively quantitative assay for comparative proteomics.Two kinds of cultured cells are grown in the medium containing either a light or heavy isotope labeled essential amino acids.After several cell doublings,the heavy amino acids are introduced into the nascent polypeptides in a sequence-specific fashion and the natural amino acid is completely replaced by its isotopically labeled analog.Accuracy quantification would be feasible by analyzing the peak intensities of every same peptide pairs labeled with light or heavy isotopes by the mass spectrometry.Compared with chemical labeling,SILAC requires much less amount of labeled proteins,and it is an efficient,straightforward,reproducible and accurate approach in cell-based systems.This method can not only investigate the dynamics of protein abundance,but also monitor the variation of posttranslational modifications and protein-protein interactions qualitatively and quantitatively.It is a powerful and important quantitative proteomics tool for tumor marker discovery.

Post-translational interaction of two fragments cleaved by protease within carboxyl-terminal domain of intestinal mucin Muc3 / 第三军医大学学报

Objective To explore the existing molecular pattern of rodent Muc3 carboxyl-terminal domain. Methods Muc3 carboxyl-terminal domain was expressed by a transient expression system and detected with SDS/PAGE and Western blotting. Identification of interaction between two proteolytically cleaved fragments was carried out by using both metabolic labeling and immunoprecipitation of expressed proteins and affinity purification of His-tagged proteins. Results Experiments with heterologous cells transfected with cDNA encoding the 381-residue C-terminal domain of rodent Muc3 showed that a definitive proteolytic cleavage occurred during the process in the endoplasmic reticulum. The products consisted of a V5-tagged 30 000 extracellular peptide, a Myc-tagged 49 000 membrane-associated peptide and non-cleaved 55 000 of whole-length protein. Two fragments remained associated by non-covalent SDS-sensitive interactions. Conclusion The proteolytic cleavage may be a prelude to later release of the large extracellular domains at cell surfaces. But the interaction between two cleaved fragments may be an important factor to interfere with the later release of the extracellular domain.