Use of dimedone-based chemical probes for sulfenic acid detection methods to visualize and identify labeled proteins.
Reversible thiol modification is a major component of the modulation of cell-signaling pathways by reactive oxygen species. Hydrogen peroxide, peroxynitrite, or lipid hydroperoxides are all able to oxidize cysteines to form cysteine sulfenic acids; this reactive intermediate can be directly reduced to thiol by cellular reductants such as thioredoxin or further participate in disulfide bond formation with glutathione or cysteine residues in the same or another protein. To identify the direct protein targets of cysteine modification and the conditions under which they are oxidized, a series of dimedone-based reagents linked to affinity or fluorescent tags have been developed that specifically alkylate and trap cysteine sulfenic acids. In this chapter, we provide detailed methods using one of our biotin-tagged reagents, DCP-Bio1, to identify and monitor proteins that are oxidized in vitro and in vivo. Using streptavidin-linked agarose beads, this biotin-linked reagent can be used to affinity capture labeled proteins. Stringent washing of the beads prior to elution minimizes the contamination of the enriched material with unlabeled proteins through coimmunoprecipitation or nonspecific binding. In particular, we suggest including DTT in one of the washes to remove proteins covalently linked to biotinylated proteins through a disulfide bond, except in cases where these linked proteins are of interest. We also provide methods for targeted approaches monitoring cysteine oxidation in individual proteins, global approaches to follow total cysteine oxidation in the cell, and guidelines for proteomic analyses to identify novel proteins with redox sensitive cysteines.
Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina, USA.
Kimberly J
KJ
Nelson
Chananat
C
Klomsiri
Simona G
SG
Codreanu
Laura
L
Soito
Daniel C
DC
Liebler
Leann C
LC
Rogers
Larry W
LW
Daniel
Leslie B
LB
Poole
D000818:Animals; D019921:Biotinylation; D003512:Cyclohexanones / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods*; D013434:Sulfenic Acids / Q000032:analysis*
D003512:Cyclohexanones; D011506:Proteins; D013434:Sulfenic Acids; C004774:dimedone