Quantification of cysteinyl S-nitrosylation by fluorescence in unbiased proteomic studies
| Autor: | Tor C. Savidge, Kizhake V. Soman, Alexander Kurosky, J. Regino Perez-Polo, Susan Stafford, Harriet C. Rea, Petri Urvil, John E. Wiktorowicz |
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| Rok vydání: | 2011 |
| Předmět: |
Nervous system
Boron Compounds Proteomics Luminescence Neurite Calorimetry Nitric Oxide Biochemistry Fluorescence Article Maleimides Random Allocation In vivo Ischemia medicine Animals Cysteine Phosphorylation Rats Wistar Hypoxia Calcium signaling Chemistry Neurogenesis S-Nitrosylation Rats Perfusion medicine.anatomical_structure Female |
| Zdroj: | Biochemistry. 50(25) |
| ISSN: | 1520-4995 |
| Popis: | Cysteinyl-S-nitrosylation has emerged as an important post-translational modification affecting protein function in health and disease. Great emphasis has been placed on global, unbiased quantification of S-nitrosylated proteins due to physiologic and oxidative stimuli. However, current strategies have been hampered by sample loss and altered protein electrophoretic mobility. Here, we describe a novel quantitative approach that combines accurate, sensitive fluorescence modification of cysteine S-nitrosylation that leaves electrophoretic mobility unaffected (SNOFlo), and introduce unique concepts for measuring changes in S-nitrosylation status relative to protein abundance. Its efficacy in defining the functional S-nitrosoproteome is demonstrated in two diverse biological applications: an in vivo rat hypoxia-ischemia reperfusion model, and antimicrobial S-nitrosoglutathione-driven transnitrosylation of an enteric microbial pathogen. The suitability of this approach for investigating endogenous S-nitrosylation is further demonstrated using Ingenuity Pathways analysis that identified nervous system and cellular development networks as the top two networks. Functional analysis of differentially S-nitrosylated proteins indicated their involvement in apoptosis, branching morphogenesis of axons, cortical neurons, and sympathetic neurites, neurogenesis, and calcium signaling. Major abundance changes were also observed for fibrillar proteins known to be stress-responsive in neurons and glia. Thus, both examples demonstrate the technique’s power in confirming the widespread involvement of S-nitrosylation in hypoxia-ischemia/reperfusion injury and in antimicrobial host responses. |
| Databáze: | OpenAIRE |
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