Quantitative analysis of redox proteome reveals oxidation-sensitive protein thiols acting in fundamental processes of developmental hematopoiesis

Proteome-wide quantitative analysis of redox cysteine availability in the Drosophila melanogaster eye reveals oxidation of phototransduction machinery during blue light exposure and age

“…Several proteomic approaches have been developed to examine cysteine availability, which can indicate its oxidation status, but many of these approaches require specialized reagents that are not commercially available [ 45 , 63 ]. We sought to characterize the redox proteome of Drosophila tissues with approaches that could be performed using readily available commercial reagents, leading us to focus on SICyLIA and iodo-TMT [ 53 , 64 ]. To identify which of these approaches would work most effectively with Drosophila samples, we compared these techniques in cultured Schneider 2 (S2) cells.…”

“…Several studies have examined redox proteomic changes in Drosophila tissues [ 56 , 57 ] and other organisms [ 45 , [48] , [49] , [50] , [51] , [52] , [53] , 64 ], but to our knowledge, this is the first study to characterize the redox proteome of either Drosophila S2 cells or the fly eye. Tissue-specific studies for redox proteomics remain relatively rare and can provide valuable insight into those proteins and pathways that are susceptible to oxidative modifications in particular cell types or tissues.…”

“…The redox proteome of mammalian cells and tissues has also been profiled using several different approaches, including an alternative method based on the iodo-TMT reagents used in our study. In this alternative approach, iodo-TMT reagents were used to identify reversibly oxidized cysteines in mouse hematopoietic stem and progenitor cells (HSPCs) comparing fetal and adult mice, and human embryonic stem cells (hESC) [ 64 ]. Reversibly oxidized cysteines such as disulfide bonds were identified by labeling free thiol groups with iodo-TMT tags, followed by a reduction step, and the addition of another iodo-TMT tag to label those thiols that has been reversibly modified [ 64 ].…”

“…In this alternative approach, iodo-TMT reagents were used to identify reversibly oxidized cysteines in mouse hematopoietic stem and progenitor cells (HSPCs) comparing fetal and adult mice, and human embryonic stem cells (hESC) [ 64 ]. Reversibly oxidized cysteines such as disulfide bonds were identified by labeling free thiol groups with iodo-TMT tags, followed by a reduction step, and the addition of another iodo-TMT tag to label those thiols that has been reversibly modified [ 64 ]. In addition, a recent study characterized the proteome of human corneal endothelial cells (iHCEC) exposed to blue light using chemoproteomic probes, which are not currently commercially available [ 63 ].…”

“…In contrast, some broad categories of proteins were reproducibly oxidized in all three models including proteins involved in translation such as eIF and ribosomal subunits. Notably, these proteins were also identified as oxidized in mammalian HSPCs [ 64 ] and mouse embryonic kidney cells treated with H 2 O 2 [ 53 ]. There were five overlapping oxidized proteins when we compared our two acute stress models, H 2 O 2 treatment in cells and prolonged blue light exposure in eyes, including eIF subunits, Hsp83, regulatory particle non-ATPase 2 (Rpn2), a regulatory subunit of the 26S proteasome, and the metabolic enzyme Ahcy.…”