No abstract

Oxidation of cysteine to sulfenic acid has emerged as a biologically relevant post-translational modification with particular importance in redox-mediated signal transduction; however, the identity of modified proteins remains largely unknown. We recently reported DAz-1, a cell-permeable chemical probe capable of detecting sulfenic acid modified proteins directly in living cells. Here we describe DAz-2, an analogue of DAz-1 that exhibits significantly improved potency in vitro and in cells. Application of this new probe for global analysis of the sulfenome in a tumor cell line identifies most known sulfenic acid modified proteins: 14 in total, plus more than 175 new candidates, with further testing confirming oxidation in several candidates. The newly identified proteins have roles in signal transduction, DNA repair, metabolism, protein synthesis, redox homeostasis, nuclear transport, vesicle trafficking, and ER quality control. Cross-comparison of these results with those from disulfide, S-glutathionylation, and S-nitrosylation proteomes reveals moderate overlap, suggesting fundamental differences in the chemical and biological basis for target specificity. The combination of selective chemical enrichment and live-cell compatibility makes DAz-2 a powerful new tool with the potential to reveal new regulatory mechanisms in signaling pathways and identify new therapeutic targets.

Photoremovable protecting groups that can reveal biologically important functional groups through one- and two-photon excitation (1PE and 2PE, respectively) have promise in regulating physiological function in a temporally and spatially restricted manner. Only a few chromophores have sufficient sensitivity to 2PE suitable for use as "caging groups" in physiology experiments. It would be useful to develop structure-property relationships of chromophores, so that chromophores with high two-photon uncaging action cross-sections (delta(u)) can be designed. The 8-bromo-7-hydroxyquinolinyl chromophore (BHQ) releases a variety of functional groups through 1PE and 2PE. Swapping the bromine substituent for a nitro (NHQ), cyano (CyHQ), or chloro (CHQ) or exchanging the hydroxy for dimethylamino (DMAQ and DMAQ-Cl) or sulfhydryl (TQ) significantly alters the photochemical and photophysical properties of the quinoline chromophore. CyHQ-OAc demonstrated a 3-fold increase in sensitivity for acetate release, whereas NHQ-OAc was photochemically insensitive. The quantum efficiencies (Q(u)) of the amino and sulfhydryl derivatives were about an order of magnitude lower than that of BHQ-OAc. All of the chromophores showed diminished sensitivity to 2PE compared to BHQ-OAc, but the CyHQ, DMAQ, and DMAQ-Cl chromophores are sufficiently sensitive for physiological use. The high sensitivity of CyHQ to 1PE will be useful in biological applications requiring short exposure with low light intensity.

Oxidation of the thiol functional group in cysteine (Cys-SH) to sulfenic (Cys-SOH), sulfinic (Cys-SO 2 H) and sulfonic acids (Cys-SO 3 H) is emerging as an important post-translational modification that can activate or deactivate the function of many proteins. Changes in thiol oxidation state have been implicated in a wide variety of cellular processes and correlate with disease states but are difficult to monitor in a physiological setting because of a lack of experimental tools. Here, we describe a method that enables live cell labeling of sulfenic acidmodified proteins. For this approach, we have synthesized the probe DAz-1, which is chemically selective for sulfenic acids and cell permeable. In addition, DAz-1 contains an azide chemical handle that can be selectively detected with phosphine reagents via the Staudinger ligation for identification, enrichment and visualization of modified proteins. Through a combination of biochemical, mass spectrometry and immunoblot approaches we characterize the reactivity of DAz-1 and highlight its utility for detecting protein sulfenic acids directly in mammalian cells. This novel method to isolate and identify sulfenic acid-modified proteins should be of widespread utility for elucidating signaling pathways and regulatory mechanisms that involve oxidation of cysteine residues.