Multiplexing protein and gene level measurements on a single Luminex platform.

BACKGROUND: The ability to simultaneously measure multiple secreted proteins and the corresponding gene expression levels from a single sample is valuable for comprehensive analysis. Bottlenecks to traditional immunoassays and gene expression assays include large sample consumption, time consuming experimental procedures, and complex data analysis. METHOD AND RESULTS: Here, we demonstrate two high-throughput assays measuring both messenger RNA (mRNA) expression and proteins in a single sample run on a Luminex platform. Human peripheral blood mononuclear cells (hPBMCs) were treated with lipopolysaccharide (LPS) and harvested at 24 and 72 h. Samples were assayed with the ProcartaPlex Human Immune Monitoring 65-plex Panel for protein and corresponding mRNA targets on a QuantiGene Human 80-plex Panel. CONCLUSION: Multiplexing ProcartaPlex and QuantiGene Plex assays provide a broad survey of protein levels and gene expression networks.

Organelle-Targeted H2S Probes Enable Visualization of the Subcellular Distribution of H2S Donors.

Hydrogen sulfide (H2S) is an essential biological signaling molecule in diverse biological regulatory pathways. To provide new chemical tools for H2S imaging, we report here a fluorescent H2S detection platform (HSN2-BG) that is compatible with subcellular localization SNAP-tag fusion protein methodologies and use appropriate fusion protein constructs to demonstrate mitochondrial and lysosomal localization. We also demonstrate the efficacy of this detection platform to image endogenous H2S in Chinese hamster ovary (CHO) cells and use the developed constructs to report on the subcellular H2S distributions provided by common H2S donor molecules AP39, ADT-OH, GYY4137, and diallyltrisulfide (DATS). The developed constructs provide a platform poised to provide new insights into the subcellular distribution of common H2S donors and a useful tool for investigating H2S biochemistry.

A Bright Fluorescent Probe for H2S Enables Analyte-Responsive, 3D Imaging in Live Zebrafish Using Light Sheet Fluorescence Microscopy.

Hydrogen sulfide (H2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H2S, is selective for H2S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H2S detection, we demonstrate the ability of MeRho-Az to detect differences in H2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H2S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems.

Hydrogen sulfide deactivates common nitrobenzofurazan-based fluorescent thiol labeling reagents.

Sulfhydryl-containing compounds, including thiols and hydrogen sulfide (H2S), play important but differential roles in biological structure and function. One major challenge in separating the biological roles of thiols and H2S is developing tools to effectively separate the reactivity of these sulfhydryl-containing compounds. To address this challenge, we report the differential responses of common electrophilic fluorescent thiol labeling reagents, including nitrobenzofurazan-based scaffolds, maleimides, alkylating agents, and electrophilic aldehydes, toward cysteine and H2S. Although H2S reacted with all of the investigated scaffolds, the photophysical response to each scaffold was significantly different. Maleimide-based, alkylating, and aldehydic thiol labeling reagents provided a diminished fluorescence response when treated with H2S. By contrast, nitrobenzofurazan-based labeling reagents were deactivated by H2S addition. Furthermore, the addition of H2S to thiol-activated nitrobenzofurazan-based reagents reduced the fluorescence signal, thus establishing the incompatibility of nitrobenzofurazan-based thiol labeling reagents in the presence of H2S. Taken together, these studies highlight the differential reactivity of thiols and H2S toward common thiol-labeling reagents and suggest that sufficient care must be taken when labeling or measuring thiols in cellular environments that produce H2S due to the potential for both false-positive and eroded responses.

Development of selective colorimetric probes for hydrogen sulfide based on nucleophilic aromatic substitution.

Hydrogen sulfide is an important biological signaling molecule and an important environmental target for detection. A major challenge in developing H2S detection methods is separating the often similar reactivity of thiols and other nucleophiles from H2S. To address this need, the nucleophilic aromatic substitution (SNAr) reaction of H2S with electron-poor aromatic electrophiles was developed as a strategy to separate H2S and thiol reactivity. Treatment of aqueous solutions of nitrobenzofurazan (7-nitro-1,2,3-benzoxadiazole, NBD) thioethers with H2S resulted in thiol extrusion and formation of nitrobenzofurazan thiol (λmax = 534 nm). This reactivity allows for unwanted thioether products to be converted to the desired nitrobenzofurazan thiol upon reaction with H2S. The scope of the reaction was investigated using a Hammett linear free energy relationship study, and the determined ρ = +0.34 is consistent with the proposed SN2Ar reaction mechanism. The efficacy of the developed probes was demonstrated in buffer and in serum with associated submicromolar detection limits as low as 190 nM (buffer) and 380 nM (serum). Furthermore, the sigmoidal response of nitrobenzofurazan electrophiles with H2S can be fit to accurately quantify H2S. The developed detection strategy offers a manifold for H2S detection that we foresee being applied in various future applications.

Synthesis of Amino-ADT Provides Access to Hydrolytically Stable Amide-Coupled Hydrogen Sulfide Releasing Drug Targets.

As additional physiological functions of hydrogen sulfide (H2S) are discovered, developing practical methods for exogenous H2S delivery is important. In particular, nonsteroidal anti-inflammatory drugs (NSAIDs) functionalized with H2S-releasing anethole dithiolethione (ADT-OH) through ester bonds are being investigated for their combined anti-inflammatory and antioxidant potential. The chemical robustness of the connection between drug and H2S-delivery components, however, is a key and controllable linkage in these compounds. Because esters are susceptible to hydrolysis, particularly under acidic conditions such as stomach acid in oral drug delivery applications, we report here a simple synthesis of amino-ADT (ADT-NH2 ) and provide conditions for successful ADT-NH2 derivatization with the drugs naproxen and valproic acid. Using UV-vis spectroscopy and HPLC analysis, we demonstrate that amide-functionalized ADT derivatives are significantly more resistant to hydrolysis than ester-functionalized ADT derivatives.

Mechanistic investigations reveal that dibromobimane extrudes sulfur from biological sulfhydryl sources other than hydrogen sulfide†Electronic supplementary information (ESI) available: Experimental details, pH stability data for BTE, NMR spectra. See DOI: 10.1039/c4sc01875cClick here for additional data file.

Hydrogen sulfide (H2S) has emerged as an important biological signaling molecule in the last decade. During the growth of this field, significant controversy has arisen centered on the physiological concentrations of H2S. Recently, a monobromobimane (mBB) method has been developed for the quantification of different biologically-relevant sulfide pools. Based on the prevalence of the mBB method for sulfide quantification, we expand on this method to report the use of dibromobimane (dBB) for sulfide quantification. Reaction of H2S with dBB results in formation of highly-fluorescent bimane thioether (BTE), which is readily quantifiable by HPLC. Additionally, the reaction of sulfide with dBB to form BTE is significantly faster than the reaction of sulfide with mBB to form sulfide dibimane. Using the dBB method, BTE levels as low as 0.6 pM can be detected. Upon use of the dBB method in wild-type and CSE-/- mice, however, dBB reports significantly higher sulfide levels than those measured using mBB. Further investigation revealed that dBB is able to extract sulfur from other sulfhydryl sources including thiols. Based on mechanistic studies, we demonstrate that dBB extracts sulfur from thiols with α- or ß-hydrogens, thus leading to higher BTE formation than from sulfide alone. Taken together, the dBB method is a highly sensitive method for H2S but is not compatible for use in studies in which other thiols are present.

Selective turn-on fluorescent probes for imaging hydrogen sulfide in living cells.

Hydrogen sulfide (H(2)S) is an important biological messenger but few biologically-compatible methods are available for its detection. Here we report two bright fluorescent probes that are selective for H(2)S over cysteine, glutathione and other reactive sulfur, nitrogen, and oxygen species. Both probes are demonstrated to detect H(2)S in live cells.