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Methodologies for the characterization, identification and quantification of S-nitrosylated proteins.

ABSTRACT: Protein S-nitrosylation plays a central role in signal transduction by nitric oxide (NO), and aberrant S-nitrosylation of specific proteins is increasingly implicated in disease.Here, methodologies for the characterization, identification and quantification of SNO-proteins are reviewed, focusing on techniques suitable for the structural characterization and absolute quantification of isolated SNO-proteins, the identification and relative quantification of SNO-proteins from complex mixtures as well as the mass spectrometry-based identification and relative quantification of sites of S-nitrosylation (SNO-sites) in proteins.Structural characterization of SNO-proteins by X-ray crystallography is increasingly being utilized to understand both the relationships between protein structure and Cys thiol reactivity as well as the consequences of S-nitrosylation on protein structure and function. New methods for the proteomic identification and quantification of SNO-proteins and SNO-sites have greatly impacted the ability to study protein S-nitrosylation in complex biological systems.The ability to identify and quantify SNO-proteins has long been rate-determining for scientific advances in the field of protein S-nitrosylation. Therefore, it is critical that investigators in the field have a good understand the utility and limitations of modern analytical techniques for SNO-protein analysis. This article is part of a Special Issue entitled: Regulation of cellular processes by S-nitrosylation.

SUBMITTER: Foster MW

PROVIDER: S-EPMC3151544 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Methodologies for the characterization, identification and quantification of S-nitrosylated proteins.

Foster Matthew W MW

Biochimica et biophysica acta 20110405 6

<h4>Background</h4>Protein S-nitrosylation plays a central role in signal transduction by nitric oxide (NO), and aberrant S-nitrosylation of specific proteins is increasingly implicated in disease.<h4>Scope of review</h4>Here, methodologies for the characterization, identification and quantification of SNO-proteins are reviewed, focusing on techniques suitable for the structural characterization and absolute quantification of isolated SNO-proteins, the identification and relative quantification ...[more]

PMID: 21440604

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Project description:Nitric oxide (NO) acts an essential signaling molecule that is involved in regulating various physiological and biochemical processes in plants. However, whether S-nitrosylation is a crucial molecular mechanism of NO is still largely unknown. In this study, 50 ?M S-nitrosoglutathione (GSNO) treatment was found to have a maximum biological effect on promoting adventitious rooting in cucumber. Meanwhile, removal of endogenous NO significantly inhibited the development of adventitious roots implying that NO is responsible for promoting the process of adventitious rooting. Moreover, application of GSNO resulted in an increase of intracellular S-nitrosothiol (SNO) levels and endogenous NO production, while decreasing the S-nitrosoglutathione reductase (GSNOR) activity during adventitious rooting, implicating that S-nitrosylation might be involved in NO-induced adventitious rooting in cucumber. Furthermore, the identification of S-nitrosylated proteins was performed utilizing the liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) and biotin-switch technique during the development of adventitious rooting. Among these proteins, the activities and S-nitrosylated level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), tubulin alpha chain (TUA), and glutathione reductase (GR) were further analyzed as NO direct targets. Our results indicated that NO might enhance the S-nitrosylation level of GAPDH and GR, and was found to subsequently reduce these activities and transcriptional levels. Conversely, S-nitrosylation of TUA increased the expression level of TUA. The results implied that S-nitrosylation of key proteins seems to regulate various pathways through differential S-nitrosylation during adventitious rooting. Collectively, these results suggest that S-nitrosylation could be involved in NO-induced adventitious rooting, and they also provide fundamental evidence for the molecular mechanism of NO signaling during adventitious rooting in cucumber explants.

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Methodologies for the characterization, identification and quantification of S-nitrosylated proteins.

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Methodologies for the characterization, identification and quantification of S-nitrosylated proteins.

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