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Development of 'Redox Arrays' for identifying novel glutathionylated proteins in the secretome.

ABSTRACT: Proteomics techniques for analysing the redox status of individual proteins in complex mixtures tend to identify the same proteins due to their high abundance. We describe here an array-based technique to identify proteins undergoing glutathionylation and apply it to the secretome and the proteome of human monocytic cells. The method is based on incorporation of biotinylated glutathione (GSH) into proteins, which can then be identified following binding to a 1000-protein antibody array. We thus identify 38 secreted and 55 intracellular glutathionylated proteins, most of which are novel candidates for glutathionylation. Two of the proteins identified in these experiments, IL-1 sRII and Lyn, were then confirmed to be susceptible to glutathionylation. Comparison of the redox array with conventional proteomic methods confirmed that the redox array is much more sensitive, and can be performed using more than 100-fold less protein than is required for methods based on mass spectrometry. The identification of novel targets of glutathionylation, particularly in the secretome where the protein concentration is much lower, shows that redox arrays can overcome some of the limitations of established redox proteomics techniques.

SUBMITTER: Mullen L

PROVIDER: S-EPMC4586893 | biostudies-other | 2015

REPOSITORIES: biostudies-other

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Development of 'Redox Arrays' for identifying novel glutathionylated proteins in the secretome.

Mullen Lisa L Seavill Miles M Hammouz Raneem R Bottazzi Barbara B Chan Philippe P Vaudry David D Ghezzi Pietro P

Scientific reports 20150929

Proteomics techniques for analysing the redox status of individual proteins in complex mixtures tend to identify the same proteins due to their high abundance. We describe here an array-based technique to identify proteins undergoing glutathionylation and apply it to the secretome and the proteome of human monocytic cells. The method is based on incorporation of biotinylated glutathione (GSH) into proteins, which can then be identified following binding to a 1000-protein antibody array. We thus ...[more]

PMID: 26416726

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Development of 'Redox Arrays' for identifying novel glutathionylated proteins in the secretome.

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Development of 'Redox Arrays' for identifying novel glutathionylated proteins in the secretome.

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