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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:Faba bean (Vicia faba L.) holds great importance for human and animal nutrition for its high protein content. However, better understanding of its seed protein composition is required in order to develop cultivars that meet market demands for plant proteins with specific quality attributes. In this study, we screened 35 diverse Vicia faba genotypes by employing the one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE) method, and 35 major protein bands obtained from three genotypes with contrasting seed protein profiles were further analyzed by mass spectrometry (MS). Twenty-five of these protein bands (MW range: ? 9-107 kDa) had significant (p ? 0.05) matches to polypeptides in protein databases. MS analysis showed that most of the analyzed protein bands contained more than one protein type and, in total, over 100 proteins were identified. These included major seed storage proteins such as legumin, vicilin, and convicilin, as well as other protein classes like lipoxygenase, heat shock proteins, sucrose-binding proteins, albumin, and defensin. Furthermore, seed protein extracts were separated by size-exclusion high-performance liquid chromatography (SE-HPLC), and percentages of the major protein classes were determined. On average, legumin and vicilin/convicilin accounted for 50 and 27% of the total protein extract, respectively. However, the proportions of these proteins varied considerably among genotypes, with the ratio of legumin:vicilin/convicilin ranging from 1:1 to 1:3. In addition, there was a significant (p < 0.01) negative correlation between the contents of these major fractions (r = -0.83). This study significantly extends the number of identified Vicia faba seed proteins and reveals new qualitative and quantitative variation in seed protein composition, filling a significant gap in the literature. Moreover, the germplasm and screening methods presented here are expected to contribute in selecting varieties with improved protein content and quality.

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

Publications

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

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