Mapping enzyme active sites in complex proteomes

Genome sequencing projects have uncovered many novel enzymes and enzyme classes for which knowledge of active site structure and mechanism is limited. To facilitate mechanistic investigations of the numerous enzymes encoded by prokaryotic and eukaryotic genomes, new methods are needed to analyze enzyme function in samples of high biocomplexity. Here, we describe a general strategy for profiling enzyme active sites in whole proteomes that utilizes activity-based chemical probes coupled with a gel-free analysis platform. We apply this gel-free strategy to identify the sites of labeling on enzymes targeted by sulfonate ester probes. For each enzyme examined, probe labeling was found to occur on a conserved active site residue, including catalytic nucleophiles (e.g., C32 in glutathione S-transferase omega) and bases/acids (e.g., E269 in aldehyde dehydrogenase-1; D204 in enoyl CoA hydratase-1), as well as residues of unknown function (e.g., D127 in 3 beta-hydroxysteroid dehydrogenase/isomerase-1). These results reveal that sulfonate ester probes are remarkably versatile activity-based profiling reagents capable of labeling a diversity of catalytic residues in a range of mechanistically distinct enzymes. More generally, the gel-free strategy described herein, by consolidating into a single step the identification of both protein targets of activity-based probes and the specific residues labeled by these reagents, provides a novel platform in which the proteomic comparison of enzymes can be accomplished in unison with a mechanistic analysis of their active sites.     

Discovering disease-associated enzymes by proteome reactivity profiling

Proteomics aims to identify new markers and targets for the diagnosis and treatment of human disease. To realize this goal, methods and reagents are needed to profile proteins based on their functional properties, rather than mere abundance. Here, we describe a general strategy for synthesizing and evaluating structurally diverse libraries of activity-based proteomic probes. Quantitative screening of probe-proteome reactions coupled with bioinformatic analysis enabled the selection of a suite of probes that exhibit complementary protein reactivity profiles. This optimal probe set was applied to discover several enzyme activities differentially expressed in lean and obese (ob/ob) mice. Interestingly, one of these enzymes, hydroxypyruvate reductase, which was 6-fold upregulated in ob/ob livers, participates in the conversion of serine to glucose, suggesting that this unusual metabolic pathway may contribute to gluconeogenesis selectively in states of obesity.     

Quantification of Bacterial Metabolic Activities in the Gut by d‐Amino Acid‐Based In Vivo Labeling

Herein, we propose a metabolic d ‐amino acid‐based labeling and in situ hybridization‐facilitated (MeDabLISH) strategy for the quantitative analysis of the indigenous metabolic status of gut bacteria. The fluorescent d ‐amino acid (FDAA)‐based labeling intensities of bacteria were found to highly correlate with their temporal and steady‐state metabolic status. Then, after taxonomic identification of bacterial genera in the in vivo FDAA‐labeled mouse gut microbiota, by corresponding fluorescence in situ hybridization (FISH) probes, the metabolic activities of different gut bacterial genera are quantified by flow cytometry, using FISH signals to differentiate genera and FDAA signals to indicate their basal metabolic levels. It was found that Gram‐negative genera in the mouse microbiota have stronger metabolic activities during the daytime, and Gram‐positive genera have higher activities at the night. Our strategy will be instrumental in deepening our understanding of the highly complex microbiota.     

Effect of oligonucleotide probes substituted by deoxyinosines on the specificity of SNP detection on the DNA microarray

One of the main factors that can affect the quality of microarray results is the microarray hybridization specificity. The key factor that affects hybridization specificity is the design of the probes. In this paper, we described a novel oligonucleotide probe containing deoxyinosines aimed at improving DNA hybridization specificity. We compared different probes to determine the distance between deoxyinosine base and SNPs site and the number of deoxyinosine bases. The new probe sequences contained two set of deoxyinosines (each set had two deoxyinosines), in which the interval between SNP site and each set of deoxyinosines was two bases. The new probes could obtain the highest hybridization specificity. The experimental results showed that probes containing deoxyinosines hybridized effectively to the perfectly matched target and improved the hybridization specificity of DNA microarray. By including a simple washing step after hybridization, these probes could distinguish matched targets from single‐base‐mismatched sequences perfectly. For the probes containing deoxyinosines, the fluorescence intensity of a match sequence was more than eight times stronger than that of a mismatch. However, the intensity ratio was only 1.3 times or less for the probes without deoxyinosines. Finally, using hybridization of the PCR product microarrays, we successfully genotyped SNP of 140 samples using these new labeled probes. Our results show that this is a useful new strategy for modifying oligonucleotide probes for use in DNA microarray analysis.     

Multicolor, one- and two-photon imaging of enzymatic activities in live cells with fluorescently Quenched Activity-Based Probes (qABPs)

Fluorescence imaging provides an indispensable way to locate and monitor biological targets within complex and dynamic intracellular environments. Of the various imaging agents currently available, small molecule-based probes provide a powerful tool for live cell imaging, primarily due to their desirable properties, including cell permeability (as a result of their smaller sizes), chemical tractability (e.g., different molecular structures/designs can be installed), and amenability to imaging a wide variety of biological events. With a few exceptions, most existing small molecule probes are however not suitable for in vivo bioimaging experiments in which high-resolution studies of enzyme activity and localization are necessary. In this article, we reported a new class of fluorescently Quenched Activity-Based Probes (qABPs) which are highly modular, and can sensitively image (through multiple enzyme turnovers leading to fluorescence signal amplification) different types of enzyme activities in live mammalian cells with good spatial and temporal resolution. We have also incorporated two-photon dyes into our modular probe design, enabling for the first time activity-based, fluorogenic two-photon imaging of enzyme activities. This, hence, expands the repertoire of 'smart', responsive probes currently available for live cell bioimaging experiments.     

Design of Optical-Imaging Probes by Screening of Diverse Substrate Libraries Directly in Disease-Tissue Extracts

Fluorescently quenched probes that are specifically activated in the cancer microenvironment have great potential application for diagnosis, early detection, and surgical guidance. These probes are often designed to target specific enzymes associated with diseases by direct optimization using single purified enzymes. However, this can result in painstaking chemistry efforts to produce a probe with suboptimal performance when applied in vivo. We describe here an alternate, unbiased activity-profiling approach in which whole tissue extracts are used to directly identify optimal peptide sequences for probe design. Screening of tumor extracts with a hybrid combinatorial substrate library (HyCoSuL) identified a combination of natural and non-natural amino-acid residues that was used to generate highly efficient tumor-specific probes. This new strategy simplifies and enhances the process of probe optimization without any a priori knowledge of enzyme targets and has the potential to be applied to diverse disease states using clinical or animal-model tissue samples.     

Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection

Understanding the complexity of the cellular environment will benefit from the ability to unambiguously resolve multiple cellular components, simultaneously and with nanometer-scale spatial resolution. Multicolor super-resolution fluorescence microscopy techniques have been developed to achieve this goal, yet challenges remain in terms of the number of targets that can be simultaneously imaged and the crosstalk between color channels. Herein, we demonstrate multicolor stochastic optical reconstruction microscopy (STORM) based on a multi-parameter detection strategy, which uses both the fluorescence activation wavelength and the emission color to discriminate between photo-activatable fluorescent probes. First, we obtained two-color super-resolution images using the near-infrared cyanine dye Alexa 750 in conjunction with a red cyanine dye Alexa 647, and quantified color crosstalk levels and image registration accuracy. Combinatorial pairing of these two switchable dyes with fluorophores which enhance photo-activation enabled multi-parameter detection of six different probes. Using this approach, we obtained six-color super-resolution fluorescence images of a model sample. The combination of multiple fluorescence detection parameters for improved fluorophore discrimination promises to substantially enhance our ability to visualize multiple cellular targets with sub-diffraction-limit resolution.     

Profiling enzyme activities in vivo using click chemistry methods

Methods for profiling the activity of enzymes in vivo are needed to understand the role that these proteins and their endogenous regulators play in physiological and pathological processes. Recently, we introduced a tag-free strategy for activity-based protein profiling (ABPP) that utilizes the copper(I)-catalyzed azide-alkyne cycloaddition reaction ("click chemistry") to analyze the functional state of enzymes in living cells and organisms. Here, we report a detailed characterization of the reaction parameters that affect click chemistry-based ABPP and identify conditions that maximize the speed, sensitivity, and bioorthogonality of this approach. Using these optimized conditions, we compare the enzyme activity profiles of living and homogenized breast cancer cells, resulting in the identification of several enzymes that are labeled by activity-based probes in situ but not in vitro.     

Confirming target engagement for reversible inhibitors in vivo by kinetically tuned activity-based probes

The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPLA1 and LYPLA2, two enzymes for which selective, in vivo active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo.     

Site-specific protein double labeling by expressed protein ligation: applications to repeat proteins

In the last few years, the use of labeled proteins has significantly expanded in the life sciences. Now, labeled proteins are indispensable tools for a wide spectrum of biophysical and chemical biology applications. In particular, the quest for more sophisticated experimental setups requires the development of new synthetic methodology, especially for multiple site-specific labeling. In this paper, we describe a synthetic strategy based on expressed protein ligation to prepare proteins in high purity and homogeneity, in which two different molecular probes are incorporated specifically at any desired position. Proteins are sequentially labeled in solution, with the advantage that a large excess of probes is not required and the labeled fragments are not restricted to peptide synthesis length limitations. This strategy was applied to selectively label a repeat protein with a fluorophores pair in different positions along the protein sequence. The doubly labeled proteins were prepared at high purity and homogeneity, as required for single molecule FRET studies. Remarkably, this approach can be adapted to the introduction of more than two molecular probes.     

Light-guided intrabodies for on-demand in situ target recognition in human cells

Due to their high stability and specificity in living cells, fluorescently labeled nanobodies are perfect probes for visualizing intracellular targets at an endogenous level. However, intrabodies bind unrestrainedly and hence may interfere with the target protein function. Here, we report a strategy to prevent premature binding through the development of photo-conditional intrabodies. Using genetic code expansion, we introduce photocaged amino acids within the nanobody-binding interface, which, after photo-activation, show instantaneous binding of target proteins with high spatiotemporal precision inside living cells. Due to the highly stable binding, light-guided intrabodies offer a versatile platform for downstream imaging and regulation of target proteins.

Nanobodies are ideal to visualize and modulate targets in living cells. We designed a versatile platform for generating photo-conditional intrabodies by genetic code expansion. After illumination, the intrabodies show fast and stable binding.     

Chemoproteomic profiling of itaconations in Salmonella

Itaconate is an immunoregulatory and anti-bacterial metabolite, and plays important roles in host–pathogen interactions. Chemoproteomic strategies have been used to explore the anti-inflammatory effects of itaconate on activated macrophages and it has been found that many key proteins in immune pathways were modified; however, how itaconate modulates pathogens was not fully understood. Here, we have designed and synthesized a series of itaconate-based bioorthogonal probes, which enable quantitative and site-specific profiling of itaconated proteins and sites in Salmonella. Among many proteins related to energy metabolism, we identified a key enzyme involved in the glyoxylate cycle, isocitrate lyase (ICL), as the most prominent target. Covalent modification of the active-site cysteine in ICL by itaconate abolishes the enzyme activity and suppresses bacterial growth. Our chemoproteomic study has uncovered the wide array of itaconation targets in Salmonella and provided a comprehensive resource for understanding the anti-bacterial function of this intriguing metabolite.

Bioorthogonal probes have been developed to enable quantitative and site-specific profiling of itaconate modifications in Salmonella.     

Enzyme shadowing: using antibody-enzyme dually-labeled magnetic particles for fast bacterial detection

Using magnetic particles and immunoseparation for target recovery and detection has been reported to improve the performance and detection limits of traditional analytical methods. For example, magnetic immunocapture can be coupled to detection in a sandwich format using an antibody (Ab) labeled with a reporter molecule or enzyme. In this work we demonstrate that simultaneous incorporation of capture and reporter biocomponents onto the sensing surface is possible and provides assays that are extremely fast and easy to carry out. As a proof of concept, we have produced dually-labeled magnetic particles, simultaneously functionalized with antibody and reporter enzyme. Subsequent capture of Escherichia coli generates a shadowing effect on the particle surface and interferes with the activity of a number of enzyme units. The decrease in signal recorded is proportional to the bacterial concentration and is specific for the target microorganism, with a detection limit of 10(3)-10(4) cell mL(-1) in an assay of about one hour.     

Profiling the specific reactivity of the proteome with non-directed activity-based probes

BACKGROUND: The field of proteomics aims to characterize dynamics in protein function on a global level. However, several classes of proteins, in particular low abundance proteins, remain difficult to characterize using standard proteomics technologies. Recently, chemical strategies have emerged that profile classes of proteins based on activity rather than quantity, thereby greatly facilitating the analysis of low abundance constituents of the proteome. RESULTS: In order to expand the classes of proteins susceptible to analysis by activity-based methods, we have synthesized a library of biotinylated sulfonate esters and applied its members to complex proteomes under conditions that distinguish patterns of specific protein reactivity. Individual sulfonates exhibited unique profiles of proteome reactivity that in extreme cases appeared nearly orthogonal to one another. A robustly labeled protein was identified as a class I aldehyde dehydrogenase and shown to be irreversibly inhibited by members of the sulfonate library. CONCLUSIONS: Through screening the proteome with a non-directed library of chemical probes, diverse patterns of protein reactivity were uncovered. These probes labeled protein targets based on properties other than abundance, circumventing one of the major challenges facing contemporary proteomics research. Considering further that the probes were found to inhibit a target enzyme's catalytic activity, the methods described herein should facilitate the identification of compounds possessing both selective proteome reactivities and novel bioactivities.     

An enzyme-based E-DNA sensor for sequence-specific detection of femtomolar DNA targets

In this work, we report an enzyme-based E-DNA sensor for the sequence-specific detection of nucleic acids. This DNA sensor employs a "stem-loop" DNA probe dually labeled with biotin and digoxigenin (DIG). The probe is immobilized at an avidin-modified electrode surface via the biotin-avidin bridge, and the DIG serves as an affinity tag for the enzyme binding. In the initial state of the sensor, the probe adopts the stem-loop structure, which shields DIG from being approached by a bulky horseradish peroxidase-linked-anti-DIG antibody (anti-DIG-HRP) due to the steric effect. After hybridization, the probe undergoes a significant conformational change, forcing DIG away from the electrode. As a result, the DIG label becomes accessible by the anti-DIG-HRP, and the target hybridization event can be sensitively transduced via the enzymatically amplified electrochemical current signal. By using this new strategy, we demonstrate that the prototype E-DNA sensor has been able to detect as low as femtomolar DNA targets with excellent differentiation ability for even single mismatches.     

A Sandwich‐Type Electrochemical Biosensor for Detection of BCR/ABL Fusion Gene Using Locked Nucleic Acids on Gold Electrode

In this study, a sandwich‐type electrochemical enzyme‐based LNA‐modified DNA biosensor was developed to detect relative gene in chronic Myelogenous Leukemia first. This biosensor is based on a ‘sandwich’ detection strategy, which involves a pair of probes (a capture probe immobilized at the electrode surface and a reporter probe labeled biotin as an affinity tag for avidin‐HRP) modified LNA. Since biotin can be connected with avidin‐HRP, this biosensor offers an enzymatically amplified electrochemical current signal for the detection of target DNA. This new pattern exhibits high sensitivity and selectivity, and this biosensor has been used for an assay of PCR real sample with satisfactory result.     

Fishing DNA targets in DNA solutions by using affinity microcontact printing

In this paper, we report the application of affinity microcontact printing (αCP) for "fishing" DNA targets in aqueous solutions and transferring them to solid surfaces for detection purposes. Affinity stamps used in this experiment were made of poly(dimethylsiloxane) (PDMS) with DNA probes covalently immobilized on their surface. When these stamps were immersed in DNA solutions, DNA targets with a perfect-match (PM) sequence to the probes can selectively hybridize to the stamp surfaces and then be transferred to solid surfaces. However, to distinguish PM DNA from single base-pair mismatch (1MM) DNA targets, 10 mM of NaCl must be added to the hybridization buffer. Under the optimized conditions, this αCP can lead to a surface density of PM which is 15 times higher than that of 1MM. The affinity stamp is also able to "fish" PM DNA targets from a mixture of PM/1MM DNA targets and transfer them to solid surfaces. Because DNA probes and targets are separated after printing, we also applied this technique for label-free detection of DNA targets by using liquid crystals.     

手术导航用荧光探针

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Interfacing Click Chemistry with Automated Oligonucleotide Synthesis for the Preparation of Fluorescent DNA Probes Containing Internal Xanthene and Cyanine Dyes

Double‐labeled oligonucleotide probes containing fluorophores interacting by energy‐transfer mechanisms are essential for modern bioanalysis, molecular diagnostics, and in vivo imaging techniques. Although bright xanthene and cyanine dyes are gaining increased prominence within these fields, little attention has thus far been paid to probes containing these dyes internally attached, a fact which is mainly due to the quite challenging synthesis of such oligonucleotide probes. Herein, by using 2′‐O‐propargyl uridine phosphoramidite and a series of xanthenes and cyanine azide derivatives, we have for the first time performed solid‐phase copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC) click labeling during the automated phosphoramidite oligonucleotide synthesis followed by postsynthetic click reactions in solution. We demonstrate that our novel strategy is rapid and efficient for the preparation of novel oligonucleotide probes containing internally positioned xanthene and cyanine dye pairs and thus represents a significant step forward for the preparation of advanced fluorescent oligonucleotide probes. Furthermore, we demonstrate that the novel xanthene and cyanine labeled probes display unusual and very promising photophysical properties resulting from energy‐transfer interactions between the fluorophores controlled by nucleic acid assembly. Potential benefits of using these novel fluorescent probes within, for example, molecular diagnostics and fluorescence microscopy include: Considerable Stokes shifts (40–110 nm), quenched fluorescence of single‐stranded probes accompanied by up to 7.7‐fold light‐up effect of emission upon target DNA/RNA binding, remarkable sensitivity to single‐nucleotide mismatches, generally high fluorescence brightness values (FB up to 26), and hence low limit of target detection values (LOD down to <5 nM ).