Use of dimedone-based chemical probes for sulfenic acid detection evaluation of conditions affecting probe incorporation into redox-sensitive proteins.
Sulfenic acids, formed as transient intermediates during the reaction of cysteine residues with peroxides, play significant roles in enzyme catalysis and regulation, and are also involved in the redox regulation of transcription factors and other signaling proteins. Therefore, interest in the identification of protein sulfenic acids has grown substantially in the past few years. Dimedone, which specifically traps sulfenic acids, has provided the basis for the synthesis of a novel group of compounds that derivatize 1,3-cyclohexadione, a dimedone analogue, with reporter tags such as biotin for affinity capture and fluorescent labels for visual detection. These reagents allow identification of the cysteine sites and proteins that are sensitive to oxidation and permit identification of the cellular conditions under which such oxidations occur. We have shown that these compounds are reactive and specific toward sulfenic acids and that the labeled proteins can be detected at high sensitivity using gel analysis or mass spectrometry. Here, we further characterize these reagents, showing that the DCP-Bio1 incorporation rates into three sulfenic acid containing proteins, papaya papain, Escherichia coli fRMsr, and the Salmonella typhimurium peroxiredoxin AhpC, are significantly different and, in the case of fRMsr, are unaffected by changes in buffer pH from 5.5 and 8.0. We also provide protocols to label protein sulfenic acids in cellular proteins, either by in situ labeling of intact cells or by labeling at the time of lysis. We show that the addition of alkylating reagents and catalase to the lysis buffer is critical in preventing the formation of sulfenic acid subsequent to cell lysis. Data presented herein also indicate that the need to standardize, as much as possible, the protein and reagent concentrations during labeling. Finally, we introduce several new test or control proteins that can be used to evaluate labeling procedures and efficiencies.
Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina, USA.
Chananat
C
Klomsiri
Kimberly J
KJ
Nelson
Erika
E
Bechtold
Laura
L
Soito
Lynnette C
LC
Johnson
W Todd
WT
Lowther
Seong-Eon
SE
Ryu
S Bruce
SB
King
Cristina M
CM
Furdui
Leslie B
LB
Poole
D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D003512:Cyclohexanones / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D008954:Models, Biological; D010084:Oxidation-Reduction; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D013434:Sulfenic Acids / Q000032:analysis* / Q000378:metabolism; D013457:Sulfur Compounds / Q000032:analysis / Q000737:chemistry
D003512:Cyclohexanones; D011506:Proteins; D013434:Sulfenic Acids; D013457:Sulfur Compounds; C004774:dimedone