Novel LC/MS/MS‐based Proteomics Approach for the Identification of S ‐Glutathionylated Proteins: Analysis of Redox Regulation of Metabolic Priming in Monocytes

We reported previously that metabolic stress primes monocytes for increased responsiveness to chemokines, a process mediated by Nox‐4‐derived H 2 O 2 and protein ‐S ‐glutathionylation. Established proteomics approaches have identified only a limited number of S ‐glutathionylated proteins. We therefore developed a novel technique using glutaredoxin to selectively reduce S ‐glutathionylated proteins, followed by labeling with a thiol‐reactive probe (iodoacetamide‐desthiobiotin), and purification using streptavidin‐coated agarose resin. We compared untreated THP‐1 monocytes with metabolically stressed (glucose, 25 mM; LDL, 100 μg/ml), or oxidatively stressed (H 2 O 2 ; 1 mM) monocytes. Using LC/MS/MS, we identified over 200 S ‐glutathionylated proteins. We also provide evidence that S ‐glutathionylation targets specific proteins for proteasome‐dependent degradation. Applying Ingenuity Systems IPA software analysis, S ‐glutathionylated proteins were associated with several cellular functions, including, protein folding and synthesis, cell motility, and cell death. In summary, we developed a novel sensitive proteomic technique for the identification of S ‐glutathionylated proteins, and provided new insights into the role of protein‐ S ‐glutathionylation in redox regulation and monocyte function in the context of metabolic disorders. Funded by NIH RO1 HL070963 & HL115858 (RA).