It combines high sensitivity with excellent linearity and fast sample turnover. Since it is free of radioactivity, it can be combined with any other modern analysis technology and can be used in high-throughput applications. Using the new method, choose size we provide for the first time an analysis of cellular fatty metabolism with high time resolution and a comprehensive comparison of utilization of a broad spectrum of fatty acids in hepatoma and adipose cell lines.
The roles of sir-2.1 in C. elegans lifespan extension have been subjects of recent public and academic debates. We applied an efficient workflow for in vivo C-13-labeling of C. elegans and C-13-heteronuclear NMR metabolomics to characterizing the metabolic phenotypes of the sir-2.1 mutant.
Our method delivered sensitivity 2 orders of magnitude higher than that of the unlabeled Inhibitors,Modulators,Libraries approach, enabling 2D and 3D NMR experiments. Multivariate analysis of the NMR data showed distinct metabolic profiles of the mutant, represented by increases in glycolysis, nitrogen catabolism, Inhibitors,Modulators,Libraries and initial lipolysis. The metabolomic analysis defined the sir-2.1 mutant metabotype as the decoupling between enhanced catabolic pathways and ATP generation. We also suggest the relationship between the metabotypes, especially the branched chain amino acids, and the roles of sir-2.1 in the worm lifespan. Our results should contribute to solidifying the roles of sir-2.1, and the described workflow can be applied to studying many other proteins in metabolic perspectives.
Many cellular factors are regulated via mechanisms affecting protein conformation, localization, and function that may be undetected by most Inhibitors,Modulators,Libraries commonly used RNA- and protein-based profiling methods that monitor steady-state gene expression. Mass-spectrometry-based chemoproteomic profiling provides alternatives for interrogating changes in the functional properties of proteins that occur in response to biological stimuli, such as viral infection. Taking dengue virus 2 (DV2) infection as a model system, we utilized reactive ATP- and ADP-acyl phosphates as chemical proteomic probes to detect changes in host kinase function that occur within the first hour of infection. The DNA-dependent protein kinase (DNA-PK) was discovered as a host enzyme with significantly elevated probe labeling within Inhibitors,Modulators,Libraries 60 min of DV2 infection.
Increased probe labeling was associated with increased DNA-PK activity in nuclear lysates and localization of DNA-PK in nucleoli. These effects on DNA-PK GSK-3 were found to require a postfusion step of DV2 entry and were recapitulated by transfection of cells with RNA corresponding to stem loop B of the DV2 5′ untranslated region. Upon investigation mostly of the potential downstream consequences of these phenomena, we detected a modest but significant reduction in the interferon response induced by DV2 in cells partially depleted of the Ku80 subunit of DNA-PK.