title: 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.
authors.affiliation: Brighton and Sussex Medical School, Trafford Centre for Medical Research, Falmer, Brighton BN1 9RY UK.
authors.forename: Lisa
authors.initials: L
authors.lastname: Mullen
authors.affiliation: Brighton and Sussex Medical School, Trafford Centre for Medical Research, Falmer, Brighton BN1 9RY UK.
authors.forename: Miles
authors.initials: M
authors.lastname: Seavill
authors.affiliation: Brighton and Sussex Medical School, Trafford Centre for Medical Research, Falmer, Brighton BN1 9RY UK.
authors.forename: Raneem
authors.initials: R
authors.lastname: Hammouz
authors.affiliation: Humanitas Clinical &Research Center Via Manzoni, 113, 20089 Rozzano, Milano, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Bottazzi
authors.affiliation: Platform in Proteomics PISSARO, Institute for Research and Innovation in Biomedicine, University of Rouen,76821 Mont-Saint-Aignan, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Chan
authors.affiliation: Platform in Proteomics PISSARO, Institute for Research and Innovation in Biomedicine, University of Rouen,76821 Mont-Saint-Aignan, France.
authors.forename: David
authors.initials: D
authors.lastname: Vaudry
authors.affiliation: Brighton and Sussex Medical School, Trafford Centre for Medical Research, Falmer, Brighton BN1 9RY UK.
authors.forename: Pietro
authors.initials: P
authors.lastname: Ghezzi
mesh_terms: D000906:Antibodies / Q000737:chemistry; D019921:Biotinylation; D002460:Cell Line; D017077:Culture Media, Conditioned / Q000737:chemistry; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D058977:Molecular Sequence Annotation; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D053574:Receptors, Interleukin-1 Type II / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019061:src-Family Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D000906:Antibodies; D017077:Culture Media, Conditioned; D008070:Lipopolysaccharides; D020543:Proteome; D053574:Receptors, Interleukin-1 Type II; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D005978:Glutathione