Versatile synthesis of probes for high-throughput enzyme activity screening

Mass spectrometry based technologies are promising as generalizable high-throughput assays for enzymatic activity. In one such technology, a specialized enzyme substrate probe is presented to a biological mixture potentially exhibiting enzymatic activity, followed by an in situ enrichment step using fluorous interactions and nanostructure-initiator mass spectrometry. This technology, known as Nimzyme, shows great potential but is limited by the need to synthesize custom substrate analogs. We describe a synthetic route that simplifies the production of these probes by fashioning their perfluorinated invariant portion as an alkylating agent. This way, a wide variety of compounds can be effectively transformed into enzyme activity probes. As a proof of principle, a chloramphenicol analog synthesized according to this methodology was used to detect chloramphenicol acetyltransferase activity in cell lysate. This verifies the validity of the synthetic strategy employed and constitutes the first reported application of Nimzyme to a non-carbohydrate-active enzyme. The simplified synthetic approach presented here may help advance the application of mass spectrometry to high-throughput enzyme activity determination.

FRET-based fluorescence probes for hydrolysis study and pig liver esterase activity

New fluorescent probes based on simple organic synthesis were designed and synthesized, and their hydrolysis catalyzed via base and pig liver esterase (PLE) was studied using FRET (fluorescence resonant energy transfer), with 1-naphthylacetic group as a donor and dansyl group as an acceptor. By simultaneous recording of changes of the donor fluorescence intensities, kinetic parameters for base-catalyzed and PLE-catalyzed hydrolysis can be determined. The presented FRET assay is a convenient and simple method and both fluorescent probes are good real-time indicators for the analysis of ester hydrolysis such as PLE activities.     

Simultaneous triggering of protein activity and fluorescence

Many areas of biology can benefit greatly from methods to spatially and temporally control protein activity. Here, we describe an approach that allows the simultaneous photo-triggering of the activity and the fluorescence of a protein. Smad2, a protein central to the transforming growth factor-beta (TGF-beta) signal transduction pathway, was modified with a fluorophore and a photocleavable moiety that acted as both a caging and a fluorescence quenching group. In its caged state, the protein formed a non-fluorescent heterodimer with the protein SARA. Irradiation with UV light and photocleavage of the caging group produced a fluorescent homotrimer. These in vitro experiments demonstrated that a photochemical trigger mimicking the critical biochemical event of serine phosphorylation involved in the TGF-beta signaling pathway could be obtained and that fluorescence could be used as a read-out of protein activity. This approach should prove particularly useful for the monitoring of a protein's activity and location inside of living cells.     

Thioamides as fluorescence quenching probes: minimalist chromophores to monitor protein dynamics

Decreasing the size of spectroscopic probes can afford higher-resolution structural information from fluorescence experiments. Therefore, we have developed p-cyanophenylalanine (Cnf) and backbone thioamides as a fluorophore/quencher pair. Through the examination of a series of thiopeptides, we have determined the working distance for this pair to be 8-30 ?. We have also carried out a proof-of-principle protein-folding experiment in which a Cnf/thioamide-labeled version of villin headpiece HP35 was thermally unfolded while the Cnf/thioamide distance was monitored by fluorescence. For a given protein, thioamide substitutions could be used to track motions with a much greater number of measurements than for current fluorescence probes, providing a dense array of data with which to model conformational changes.     

Interrogating signaling nodes involved in cellular transformations using kinase activity probes

Protein kinases catalyze protein phosphorylation and thereby control the flow of information through signaling cascades. Currently available methods for concomitant assessment of the enzymatic activities of multiple kinases in complex biological samples rely on indirect proxies for enzymatic activity, such as posttranslational modifications to protein kinases. Our laboratories have recently described a method for directly quantifying the enzymatic activity of kinases in unfractionated cell lysates using substrates containing a phosphorylation-sensitive unnatural amino acid termed CSox, which can be monitored using fluorescence. Here, we demonstrate the utility of this method using a probe set encompassing p38α, MK2, ERK1/2, Akt, and PKA. This panel of chemosensors provides activity measurements of individual kinases in a model of skeletal muscle differentiation and can be readily used to generate individualized kinase activity profiles for tissue samples from clinical cancer patients.     

Polymeric nanoparticles for protein kinase activity

Nanoparticles were prepared from poly-ion complexes, possessing both PEI-FITC-(PKA-specific substrate) (kemptide) and PAA-TRITC, which produce intermolecular FRET; the nanoparticles were dissociated by phosphorylation, presented a strong FITC intensity and can be applied to high-throughput screening for large chemical libraries, for drug discovery of kinase inhibitors.     

Free-probe fluorescence of light-up probes.

The fluorescence enhancement of light-up probes (thiazole orange (TO) conjugated peptide nucleic acids (PNAs)) upon hybridization to target nucleic acid depends on the probe sequence, mainly due to large variations in free-probe fluorescence. Here we study three probes where the fluorescence in free state varies more than 50-fold. We find that this variation is due to a fraction that has TO intramolecularly "back-bound" to the PNA bases. The intramolecular affinity constant for this unimolecular interaction was determined by temperature titrations using absorption spectroscopy, and the fluorescence quantum yields of the probes in back-bound conformation were calculated. The molar ratio of probes in back-bound conformation was 0.70-0.96 at 30 degrees C and 0.40-0.73 at 60 degrees C, and the fluorescence quantum yield in back-bound conformation varied between 0.0020 and 0.077 at 30 degrees C, and 0.00065-0.029 at 60 degrees C. These data show that the variation in free-probe fluorescence depends mainly on the fluorescence quantum yield of the probe in back-bound conformation and to a much lesser extent on the tendency of the probe to adopt the back-bound conformation. With increasing temperature the free-probe fluorescence decreases owing to both reduced degree of back-binding and a decrease of the fluorescence quantum yield in back-bound conformation.     

Light-regulated sampling of protein tyrosine kinase activity

Protein tyrosine kinases serve as key mediators of signaling pathways, biochemical highways that control various aspects of cell behavior. Although fluorescent reporters of tyrosine kinases have been described, these species can suffer immediate phosphorylation upon exposure to the cellular milieu, thereby hindering a detailed analysis of kinase activity as a function of the cell cycle or exposure to environmental stimuli. The first example of a light-regulated tyrosine kinase reporter is described herein, which allows the investigator to control when kinase activity is sampled. In addition, the set of sensors created in this study contain different fluorophores, each with its own unique photophysical properties, thereby furnishing an array of choices that can be used in combination with other intracellular probes.     

Near-infrared fluorescence probes for surgical navigation

Optical imaging is a promising tool for visualizing fundamental biological processes including disease progression, detection of tumors, and therapeutic monitoring non-invasively. Unlike visible light, near-infrared fluorescence (NIRF) imaging (beyond 700–1,700 nm) offers a competitive advantage to yield high-resolution images within a certain penetration depth (few millimeters to centimeters depending on NIR window). The last few years have witnessed rapid development of new NIRF probes within the span of whole NIR window, including small-molecule dyes, inorganic nanoparticles, and organic macromolecules. Benefitted by this, we observe a continual surge in the number of preclinical and clinical studies of NIRF imaging in surgery and related applications. At present, NIRF-guided imaging has emerged as a quintessential procedure to assist surgeons for intraoperative delineation and resection of tumors. Moreover, NIRF imaging is also used to improve the intraoperative staging, identify the hidden lesion in diseased organs, map lymph node metastases, detect tumor margins, and highlight vital organs intraoperatively. Considering rapid advancement of this field, we review recent progress in the development of NIRF probes, cancer-targeting strategies and their application for surgical navigation, particularly for the sentinel lymph node mapping, detection of tumors, and angiography. Moreover, we spotlight surgical navigation instrumentation that is currently used for intraoperative tumor detection.     

Anthryl-substituted heterocycles as acid-sensitive fluorescence probes

[reaction: see text] Four pH-sensitive fluorescence probes are presented which consist of an anthracene fluorophore and a pi-conjugated oxazoline, benzoxazole, or pyridine substituent. The protonation of the heterocycles increases their acceptor properties and results in significant red-shifts of the absorption and emission maxima of the anthracene chromophore. The comparison between 2-[2'-(6'-methoxyanthryl)]-4,4-dimethyl-2-oxazoline and 2-[2'-(anthryl)]-4,4-dimethyl-2-oxazoline reveals that the donor-acceptor substitution pattern of the fluorophore is not required to achieve a red shift upon protonation. The benzoxazole and pyridine substituents offer a particular advantage due to their persistence under acidic conditions. Thus, these compounds may be used as efficient pH-sensitive fluorescence switches. Nevertheless, the switching of benzoxazole 2c requires relatively strong acidic conditions. The anthrylpyridinium exhibits a red-shifted emission in chloroform; however, it is nonfluorescent in aqueous or alcoholic solution. Although the oxazoline is not persistent under permanent acidic conditions, this heterocycle may be useful as a substituent in fluorescence indicators since it may be used to detect acid concentrations of 10(-4)-10(-5) M, which are close to the biologically relevant range.     

A light-activated probe of intracellular protein kinase activity

The first example of a photoactivated probe of intracellular enzymatic activity is described. The caged derivative of a fluorescent protein kinase C peptide-based sensor was prepared by modifying the free hydroxyl group of a phosphorylatable serine moiety with a photolabile appendage that blocks phosphoryl transfer. We have demonstrated that the caged sensor allows one to (1) sample PKC activity with exquisite temporal precision, (2) control the relative amount of active sensor available for phosphorylation, and (3) examine protein kinase activity at multiple time points.     

Green/red dual fluorescence detection of total protein and alkaline phosphate-conjugated probes on blotting membranes

A two-color fluorescence detection method is described based upon covalently coupling the succinimidyl ester of BODIPY FL-X to proteins immobilized on poly(vinylidene difluoride) (PVDF) membranes, followed by detection of target proteins using the fluorogenic substrate 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl(DDAO)-phosphate in combination with alkaline-phosphatase-conjugated reporter molecules. This results in all proteins in the profile being visualized as green signal while those detected specifically with the alkaline-phosphatase conjugate appear as red signal. The dichromatic detection system is broadly compatible with a wide range of analytical imaging devices including UV epi- or transilluminators combined with photographic or charge-coupled device (CCD) cameras, xenon-arc sources equipped with appropriate excitation/emission filters, and dual laser gel scanners outfitted with a 473 nm second-harmonic generation or 488 nm argon-ion laser as well as a 633 nm helium-neon or 635 nm diode laser. The dichromatic detection method permits detection of low nanogram amounts of protein and allows for unambiguous identification of target proteins relative to the entire protein profile on a single electroblot, obviating the need to run replicate gels that would otherwise require visualization of total proteins by silver staining and subsequent alignment with chemiluminescent or colorimetric signals generated on electroblots.     

ADP-specific sensors enable universal assay of protein kinase activity

Two molecular sensors that specifically recognize ADP in a background of over 100-fold molar excess of ATP are described. These sensors are nucleic-acid based and comprise a general method for monitoring protein kinase activity. The ADP-aptamer scintillation proximity assay is configured in a single-step, homogeneous format while the allosteric ribozyme (RiboReporter) sensor generates a fluorescent signal upon ADP-dependent ribozyme self-cleavage. Both systems perform well when configured for high-throughput screening and have been used to rediscover a known protein kinase inhibitor in a high-throughput screening format.     

Competitive fluorescence polarization assays for the detection of phosphoinositide kinase and phosphatase activity

We describe the development and implementation of competitive fluorescence polarization (FP) based assays for determining activity of phosphoinositide 3-kinase (PI 3-K) and the type-II SH2-domain-containing inositol 5-phosphatase (SHIP2). These assays are based on the interaction of specific phosphoinositide binding proteins with fluorophore-labeled phosphoinositide and inositol phosphate tracers. Enzyme reaction products are detected by their ability to compete with the fluorescent tracers for protein binding, leading to an increase in the amount of free tracer and a decrease in polarization (mP) values. A variety of fluorophore-labeled tracers were evaluated, and assay sensitivity and specificity for products of PI 3-K and SHIP2 activity was determined. Assay performance was evaluated using recombinant PI 3-Kalpha and SHIP2 with diC(8)-PI(4,5)P(2) and diC(8)-PI(3,4,5)P(3) as respective substrates. IC(50) values for previously characterized PI 3-K inhibitors were within expected ranges. These assays are homogeneous, sensitive, and rapid, and suitable for HTS applications, and will facilitate screening for novel inhibitors of phosphoinositide kinases and phosphatases in drug development.     

Label free fluorescence turn-on detection of polynucleotide kinase activity with a perylene probe

A single stranded oligonucleotide could induce aggregation of a perylene probe, the probe's monomer fluorescence was efficiently quenched. However, when the oligonucleotide was 5'-phosphorylated by polynucleotide kinase, it could be very efficiently degraded by lambda exonuclease, probe monomers were released, and a turn on fluorescence signal was detected.     

Intracellular pH measurements made simple by fluorescent protein probes and the phasor approach to fluorescence lifetime imaging

A versatile pH-dependent fluorescent protein was applied to intracellular pH measurements by means of the phasor approach to fluorescence lifetime imaging. By this fit-less method we obtain intracellular pH maps under resting or altered physiological conditions by single-photon confocal or two-photon microscopy.     

Versatile glycoblotting nanoparticles for high-throughput protein glycomics

We have developed an effective and practical trap-and-release method based on chemoselective ligation of carbohydrates with reactive aminooxyl groups attached to the surface of nanoparticles (referred to as glycoblotting nanoparticles). These glycoblotting nanoparticles were synthesized by UV irradiation of diacetylene-functionalized lipids that contain the aminooxyl group. The glycoblotting nanoparticles captured carbohydrates in aqueous solution under mild conditions and were collected by simple centrifugation. The trapped carbohydrates were effectively released from the nanoparticles under acidic conditions to give pure oligosaccharides. This glycoblotting process reduced the time required for the purification process of carbohydrates to less than 6 h, compared to the several days needed for conventional chromatographic techniques. The oligosaccharides (N-glycan) were released from ovalbumin (glycoprotein) by PNGase F after tryptic digestion. MALDI-TOF mass spectra before purification did not show any significant signals corresponding to N-glycans because these signals were hidden by the large signals of the abundant peptides. However, after purification with the glycoblotting nanoparticles, only signals corresponding to oligosaccharides appeared. We also demonstrated a clear analysis of the oligosaccharides contained in the mice dermis by means of glycoblotting.     

Label-free fluorescence detection of the depurination activity of ribosome inactivating protein toxins

Development of a simple label-free fluorescence hybridization assay to monitor the depurination activity of toxic ribosome inactivating proteins by using a fluorescent ligand that specifically pseudo base pairs with a cytosine residue opposite an abasic site is described. This method could be potentially implemented in screening platforms for the discovery of small molecules that inhibit the activity of these toxins.