Synthesis and Use of the Bifunctional Sulfenic Acid Probe BCN‐E‐BCN for In Vitro and Cell‐Based Assays of Protein Oxidation

Abstract

The reversible oxidation of cysteine thiol groups to sulfenic acid by reactive oxygen species (ROS) such as hydrogen peroxide can impact protein function, activity, and localization. As a consequence, ROS have profound effects on cell functions including proliferation, differentiation, and survival. Furthermore, there are clear associations between the effects of ROS on cells and the etiology of several diseases including cancer and neurodegeneration. In spite of the importance of cysteine sulfenylation as a validated post‐translational modification, its labile nature impedes efficient and reproducible detection of proteins with cysteine sulfenic acid residues. To overcome this challenge, we developed a novel cell‐permeable bifunctional reagent, consisting of two linked bicyclo[6.1.0]nonyne (BCN) moieties coupled with a short ethylenediamine‐derived linker (BCN‐E‐BCN) that enables the detection of sulfenylated proteins in vitro and in intact cells. The two symmetrical BCN groups allow protein sulfenic acids to be selectively tagged with a BCN at one end while allowing for copper‐free click chemistry with azide‐tagged reagents of the opposite BCN. In this protocol, the synthesis of BCN‐E‐BCN and its use to detect cysteine sulfenic acids will be detailed. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Copper‐mediated cyclopropanation of 1,5‐cyclooctadiene Basic Protocol 2: Synthesis of endo‐ and exo‐bicyclononyne Basic Protocol 3: Synthesis of endo‐BCN‐E‐BCN Basic Protocol 4: BCN‐E‐BCN treatment of wild‐type and cysteine‐deficient mutant recombinant cofilin protein Basic Protocol 5: BCN‐E‐BCN labeling in live cells Basic Protocol 6: Western blotting and visualization of BCN‐E‐BCN‐labeled samples

S-Sulfenylation Driven Antigen Capture Boosted by Radiation for Enhanced Cancer Immunotherapy

“…Exposure of redox-sensitive cysteine residues to ROS (e.g., H 2 O 2 ) eventually results in the specific oxidation of cysteine thiols of target proteins to form sulfenic acids. 30,31 The numbers of proteins captured by conventional maleimide-decorated AuNPs (Mal@AuNPs) were slightly higher than the ones captured by dAuNPs we used, whereas more sulfenylated proteins should be essentially captured by our method, and they are more likely to be tumor antigens. More importantly, many of these proteins were demonstrated to be closely associated with antigen processing-cross presentation as well as innate immunity.…”

“…Ionizing radiation is a well-known producer of oxidative stress that can generate vast amounts of reactive oxygen species (ROS) through the radiolysis of water, including hydroxyl radicals ( • OH), alkoxyl radicals, superoxide anion (O 2 •– ), singlet oxygen ( 1 O 2 ), hydrogen peroxide (H 2 O 2 ), etc . The sulfhydryl groups of TDPAs can be simultaneously oxidized to sulfenic (Cys-SOH), sulfinic (Cys-SO 2 H), or sulfonic (Cys-SO 3 H) groups. − Hence, the S -sulfenylation degree of proteins is often used to evaluate the pathological course of tumors based on the characterization of high ROS levels in the tumor microenvironment. , Since protein S -sulfenylation (PSS) has been reported to be closely associated with the activation, proliferation, and function of CD8 T + cells, we hypothesized that the specific and efficient capture of the sulfenylated antigens by functional nanoparticles may induce strong immunogenicity for immune response activation, which could be highly meaningful for improving the immunotherapeutic efficacy of cancer treatment as well as the abscopal effect.…”