Probe Labeling Research Articles

Fluorogenic Labeling Probe for the Imaging of Endogenous β-Galactosidase Activity in Cancer and Senescent Cells.

The sensitive detection of glycosidases in live cells is crucial to understanding their functional roles in disease progression. Here, we develop a fluorogenic labeling probe for β-galactosidase (β-Gal) based on a bright green-emitting fluorescent dye, fluorescein. Galactose was introduced to a fluoromethyl-substituted fluorescein derivative through a benzyl spacer, resulting in a quenched fluorescence due to spirocyclization of the dye. After removal of the galactosyl residue by β-Gal, an ∼210-fold enhanced green fluorescence (emission maximum at 524 nm) was detected, and the presence of other glycosidases and hydrolases did not produce false-positive signals. The probe was successfully used for imaging of the endogenous β-Gal activity in cancer and senescent cells, and the imaging results agree with the β-Gal expression level of the cells, as determined by Western blotting and polymerase chain reaction. Importantly, we demonstrated that upon hydrolysis of galactose, the fluoromethyl-substituted fluorescein derivative is covalently attached to adjacent proteins, both in solution and in live cells. This study offers a small-molecule probe for the sensitive monitoring of endogenous glycosidase activity.

In vivo scanning laser fundus and high-resolution OCT imaging of retinal ganglion cell injury in a non-human primate model with an activatable fluorescent-labeled TAT peptide probe.

The optical imaging agent TcapQ488 has enabled imaging of retinal ganglion cell (RGC) injury in vivo in rodents and has potential as an effective diagnostic probe for early detection and intervention monitoring in glaucoma patients. In the present study, we investigated TcapQ488 in non-human primates (NHPs) to identify labeling efficacy and early signals of injured RGC, to determine species-dependent changes in RGC probe uptake and clearance, and to determine dose-limiting toxicities. Doses of 3, 6, and 12 nmol of TcapQ488 were delivered intravitreally to normal healthy NHP eyes and eyes that had undergone hemiretinal endodiathermy axotomy (HEA) in the inferior retina. Post-injection fundus fluorescence imaging using a Spectralis imaging platform (Heidelberg Engineering) documented TcapQ488 activation in RGC cell bodies. Optical coherence tomography (OCT), slit-lamp examinations, intraocular pressure measurements, and visual electrophysiology testing were performed to monitor probe tolerability. For comparison, a negative control, non-cleavable, non-quenched probe (dTcap488, 6 nmol), was delivered intravitreally to a normal healthy eye. In normal healthy eyes, intravitreal injection of 3 nmol of TcapQ488 was well-tolerated, while 12 nmol of TcapQ488 to the healthy eye caused extensive probe activation in the ganglion cell layer (GCL) and eventual retinal nerve fiber layer thinning. In HEA eyes, the HEA procedure followed by intravitreal TcapQ488 (3 nmol) injection resulted in probe activation within cell bodies in the GCL, confined to the HEA-treated inferior retina, indicating cell injury and slow axonal transport in the GCL. However, in contrast to rodents, a vitreal haze that lasted 2-12 weeks obscured rapid high-resolution imaging of the fundus. By contrast, intravitreal TcapQ488 injection prior to the HEA procedure led to minimal probe labeling in the GCL. The results of the dTcap488 control experiments indicated that fast axonal transport carried the probe out of the retina after cell body uptake. No evidence of pan-retinal toxicity or loss of retino-cortical function was detected in any of the three NHPs tested. Overall, these data provide evidence of TcapQ488 activation, without toxicity, in NHP HEA eyes that had been intravitreally injected with 3 nmol of the probe. Compared to rodents, unexpectedly rapid axonal transport in the NHPs reduced the capacity to visualize RGC cell bodies and axons through the backdrop of an intravitreal haze. Nonetheless, although intravitreal clearance rates did not scale to NHPs, HEA-induced reductions in axonal transport enhanced probe visualization in the cell body.

A Long Fluorescence Lifetime Probe for Labeling of Gram-Negative Bacteria

A Long Fluorescence Lifetime Probe for Labeling of Gram-Negative Bacteria

Genetically encoded intrabody probes for labeling and manipulating AMPA-type glutamate receptors

Tools for visualizing and manipulating protein dynamics in living cells are critical for understanding cellular function. Here we leverage recently available monoclonal antibody sequences to generate a set of affinity tags for labeling and manipulating AMPA-type glutamate receptors (AMPARs), which mediate nearly all excitatory neurotransmission in the central nervous system. These antibodies can be produced from heterologous cells for exogenous labeling applications or directly expressed in living neurons as intrabodies, where they bind their epitopes in the endoplasmic reticulum and co-traffic to the cell surface for visualization with cell impermeant fluorescent dyes. We show these reagents do not perturb AMPAR trafficking, function, mobility, or synaptic recruitment during plasticity and therefore can be used as probes for monitoring endogenous receptors in living neurons. We also adapt these reagents to deplete AMPARs from the cell surface by trapping them in the endoplasmic reticulum, providing a simple approach for loss of excitatory neurotransmission. The strategies outlined here serve as a template for generating similar reagents targeting diverse proteins as more antibody sequences become available.

Tunable Activated Esters Enable Lysine-Selective Protein Labeling and Profiling.

Lysine residues on protein surfaces are abundant and often found in enzyme active sites, making them critical targets for studying undruggable proteins. However, the varied microenvironment surrounding lysine residues results in a wide range of pKa values, complicating site-specific covalent binding. In this study, we address the challenges posed by the diverse reactivity of amino side chains by modulating the amide reaction activity of heteroaromatic activated esters. By fine-tuning the type, position, and number of heteroatoms, we successfully rationalized the regulation of their amide reaction activity, leading to the design of probes for selective lysine labeling within the proteome for profiling purposes. Systematic optimization of these esters' reactivity and selectivity has yielded a series of effective probes suitable for both in vitro and cellular applications. These findings significantly enhance our understanding of protein functions and mechanisms, facilitated by the precise identification and analysis of protein labeling and profiling.

Ac34deoGlcNAz: A Selective Probe for Identifying O-GlcNAc-Modified Proteins in Prostate Cancer.

O-GlcNAcylation is a prevalent protein modification in eukaryotic cellsandincreasing evidences indicated that over-expressed O-GlcNAcylation are intimately linked to the development and prognosis of prostate cancer.Thus, exploring this modification in the context of prostate cancer is vital for understanding the underlying mechanisms and hopefully used forfuture targeted therapies. In this paper, we use our previously established metabolic probes to comprehensively compare the labeling efficiency ofO-GlcNAc modified proteins in PC3 cells. Our results demonstrated that all the tested probes were non-toxic to PC3 cells and only Ac4GlcNAz, Ac4GalNAzand Ac34deoGlcNAzexhibited robust labeling signals amongst the probes. Further investigations by western blot and flow cytometry analysis revealed that Ac34deoGlcNAzwas a specific and efficient probe for intracelluar protein labeling with negligible S-glyco-modification signal. In addition, proteomic analysis further confirmed that 94% of the proteins identified byAc34deoGlcNAzwere in the form of O-linked GlcNAc, these enriched O-GlcNAcylated proteins were mainly involved in the regulated processes of prostate cancer. Our results here together prove Ac34deoGlcNAzis a safe and reliable probefor metabolic labeling O-GlcNAc modified proteins in prostate cancer, providing a mean to fully exploitthe regulatory mechanism of O-GlcNAcylation in the process of prostate cancer.

Rapid screening of prednisolone acetate adulterants in health foods using colloidal gold immunochromatographic assay

In this study, a rapid detection method utilizing colloidal gold immunochromatography (CG-ICA) was developed for the detection of illegally added prednisone acetate in health foods. Initially, the preparation conditions of colloidal gold solution were optimized. The optimal potassium carbonate dosage, antibody diluent type, antibody dosage, probe labeling time, blocking time and BSA dosage were determined. Technical analysis was performed to ensure that the established CG-ICA exhibited satisfactory color development and inhibition rates. Under optimized conditions, the cut-off value of CG-ICA was 250 μg/kg. The assay demonstrated a sensitivity of 100 %, a false positive rate of 8 %, and a false negative rate of 0, indicating high specificity for prednisone acetate. The results obtained from testing actual samples were consistent with those obtained using LC-MS/MS, thereby verifying the reliability of the developed method. This method offers robust support for the rapid detection of illegally added prednisone acetate in health foods.

Fluorescent labeling of live-cell surfaceome and its application in antibody-target interaction analysis

BackgroundCell-surface proteins play key roles in the communication between external stimuli and internal signaling. As protein types and expression levels vary in different cells, in-situ visualization of the whole surface proteome (surfaceome) may facilitate the study of their functions in homeostasis maintenance or response to environmental changes (e.g., drug treatment). However, there lacks easily-prepared and universal labeling probes to visualize them in living cells. ResultsWe designed and synthesized a small-molecule fluorescent probe, SRB-NHS, for one-step labeling of surfaceome. Live-cell imaging results exhibited the plasma membrane localization of the fluorescent signal from SRB-NHS and SDS-PAGE/fluorescence scanning results confirmed the covalent labeling of proteins by SRB-NHS, indicating the suitability of SRB-NHS for surfaceome labeling towards different cell lines. SignificanceUpon labeling by SRB-NHS, the cellular internalization of surfaceome was studied under different stimuli (e.g., nutritional deprivation, drug treatments). Intriguingly, specific monitoring of the interaction between antibody drugs and related cell-surface targets can be achieved when the probe is used in combination with fluorescently labeled antibodies and imaged via Förster resonance energy transfer (FRET), offering a new method compatible with various cell lines to monitor the surfaceome or a specific drug-target interaction in situ.

Hippocampal γCaMKII dopaminylation promotes synaptic-to-nuclear signaling and memory formation.

Protein monoaminylation is a class of posttranslational modification (PTM) that contributes to transcription, physiology and behavior. While recent analyses have focused on histones as critical substrates of monoaminylation, the broader repertoire of monoaminylated proteins in brain remains unclear. Here, we report the development/implementation of a chemical probe for the bioorthogonal labeling, enrichment and proteomics-based detection of dopaminylated proteins in brain. We identified 1,557 dopaminylated proteins - many synaptic - including γCaMKII, which mediates Ca2+-dependent cellular signaling and hippocampal-dependent memory. We found that γCaMKII dopaminylation is largely synaptic and mediates synaptic-to-nuclear signaling, neuronal gene expression and intrinsic excitability, and contextual memory. These results indicate a critical role for synaptic dopaminylation in adaptive brain plasticity, and may suggest roles for these phenomena in pathologies associated with altered monoaminergic signaling.

Engineering of exosome-liposome hybrid-based theranostic nanomedicines for NIR-II fluorescence imaging-guided and targeted NIR-II photothermal therapy of subcutaneous glioblastoma

Exosome-liposome hybrid-based vehicles (ELV) are promising carriers for cancer treatment, but there are rare efficient theranostic probes to label their lipid bilayer membrane for precisely tracing biodistribution and execute potent therapy. As both fluorescence imaging and photothermal therapy in the second near-infrared window (NIR-II) has intrinsically deep penetration and high efficacy to ablate tumors, herein the design and synthesis of lipophilic NIR-II cyanine dyes with strong donor strength is reported to label lipid bilayer membrane of ELV for NIR-II fluorescence image-guided and targeted NIR-II photothermal treatment of subcutaneous glioblastoma. Via lipid film hydration and subsequent extrusion method, the synthesized ELV (NIR-C12-EL) is formulated with NIR-C12 labeling, cyclic arginylglycylaspartic acid decoration, liposomal PEGylation, and biological exosome function. NIR-C12-EL exhibits excellent colloidal stability, good biocompatibility, strong light harvesting capability, high NIR-II photoconversion efficiency (62.28 %), and targeting capability to diagnose and ablate tumors, which together contribute to the extended life-span of the mice treatment with NIR-C12-EL and continuous 1064 nm laser irradiation. This study provides insight into not only designing of lipophilic NIR-II fluorescence probes for labeling of exosome-liposome hybrid-based vehicles but also the engineering of theranostic nanoplatforms for precise treatment of glioblastoma.

Isotope-Labeled Chemoselective Probes for Labeling, Separation, and Comprehensive Quantitative Analysis of Sub-Metabolome.

The significance of small molecule metabolites as biomarkers for disease diagnosis and prognosis is growing increasingly evident, necessitating the development of highly sensitive qualitative and quantitative methods. Herein, multi-chemoselective probes are synthesized and applied for profiling metabolites, including carboxyl, phosphate, hydroxyl, amino, thiol, and carbonyl compounds. This approach seamlessly integrates magnetic solid-phase materials, orthogonal cleavage sites, isotopic tags, and selective coupling sites, minimizes matrix interference, and enhances quantitative accuracy. Meanwhile, a homemade program, High-Resolution Isotope-Assisted Identification and Quantitative (HRIAIQuant) is developed to process the data, which adeptly filters through 33,874 ion pairs present in human serum, leading to the identification of 701 known metabolites and a remarkable 1,062 potential novel ones. This method is successfully applied to analyze metabolites in multiple brain regions of SAMP8 and SAMR1 models, offering a novel tool for Alzheimer's disease research.

Profiling sorghum-microbe interactions with a specialized photoaffinity probe identifies key sorgoleone binders in Acinetobacter pittii.

Interactions between plants and soil microbial communities that benefit plant growth and enhance nutrient acquisition are driven by the selective release of metabolites from plant roots, or root exudation. To investigate these plant-microbe interactions, we developed a photoaffinity probe based on sorgoleone (sorgoleone diazirine alkyne for photoaffinity labeling, SoDA-PAL), a hydrophobic secondary metabolite and allelochemical produced in Sorghum bicolor root exudates. We applied SoDA-PAL to the identification of sorgoleone-binding proteins in Acinetobacter pittii SO1, a potential plant growth-promoting microbe isolated from sorghum rhizosphere soil. Competitive photoaffinity labeling of A. pittii whole cell lysates with SoDA-PAL identified 137 statistically enriched proteins, including putative transporters, transcriptional regulators, and a subset of proteins with predicted enzymatic functions. We performed computational protein modeling and docking with sorgoleone to prioritize candidates for experimental validation and then confirmed binding of sorgoleone to four of these proteins in vitro: the α/β fold hydrolase SrgB (OH685_09420), a fumarylacetoacetase (OH685_02300), a lysophospholipase (OH685_14215), and an unannotated hypothetical protein (OH685_18625). Our application of this specialized sorgoleone-based probe coupled with structural bioinformatics streamlines the identification of microbial proteins involved in metabolite recognition, metabolism, and toxicity, widening our understanding of the range of cellular pathways that can be affected by a plant secondary metabolite.IMPORTANCEHere, we demonstrate that a photoaffinity-based chemical probe modeled after sorgoleone, an important secondary metabolite released by sorghum roots, can be used to identify microbial proteins that directly interact with sorgoleone. We applied this probe to the sorghum-associated bacterium Acinetobacter pittii and showed that probe labeling is dose-dependent and sensitive to competition with purified sorgoleone. Coupling the probe with proteomics and computational analysis facilitated the identification of putative sorgoleone binders, including a protein implicated in a conserved pathway essential for sorgoleone catabolism. We anticipate that discoveries seeded by this workflow will expand our understanding of the molecular mechanisms by which specific metabolites in root exudates shape the sorghum rhizosphere microbiome.

Development of a monomolecular multifunctional imaging probe for dual labeling of LDL to image and characterize atherosclerotic plaques

Development of a monomolecular multifunctional imaging probe for dual labeling of LDL to image and characterize atherosclerotic plaques

Chemoproteomic Strategy Identifies PfUCHL3 as the Target of Halofuginone.

The human malaria parasite Plasmodium falciparum (P. falciparum) continues to pose a significant public health challenge, leading to millions of fatalities globally. Halofuginone (HF) has shown a significant anti-P. falciparum effect, suggesting its potential as a therapeutic agent for malaria treatment. In this study, we synthesized a photoaffinity labeling probe of HF to identify its direct target in P. falciparum. Our results reveal that ubiquitin carboxyl-terminal hydrolase 3 (PfUCHL3) acts as a crucial target protein of HF, which modulates parasite growth in the intraerythrocytic cycle. In particular, we discovered that HF potentially forms hydrogen bonds with the Leu10, Glu11, and Arg217 sites of PfUCHL3, thereby inducing an allosteric effect by promoting the embedding of the helix 6' region on the protein surface. Furthermore, HF disrupts the expression of multiple functional proteins mediated by PfUCHL3, specifically those that play crucial roles in amino acid biosynthesis and metabolism in P. falciparum. Taken together, this study highlights PfUCHL3 as a previously undisclosed druggable target of HF, which contributes to the development of novel anti-malarial agents in the future.

Labeling selectivity of lipid droplets fluorescent probes: Twisted intramolecular charge transfer (TICT) vs intramolecular charge transfer (ICT)

Fluorescence imaging technology is a versatile and essential tool in the field of biomedical research. To obtain excellent imaging results, the precise labeling of fluorescent probes is an important prerequisite. Nevertheless, the labeling selectivity of most fluorescent probes is not satisfactory, new design concepts are desperately needed. In this context, two isomeric lipid droplets (LDs) fluorescent probes Lipi-Cz-1 and Lipi-Cz-2 have been sophisticatedly developed with TICT and ICT-emitting characteristic, respectively. The more environmentally sensitive TICT-emitting Lipi-Cz-1 exhibits a significantly enhanced labeling selectivity in LDs imaging compared to the ICT-emitting Lipi-Cz-2, sufficiently illustrating the effectiveness of TICT-emitting characteristic in improving labeling selectivity. Additionally, Lipi-Cz-1 displays high photostability and biocompatibility. These advantages enable Lipi-Cz-1 to be finely applied in multimode fluorescence imaging, e.g. time-lapse 3D confocal imaging to monitor changes of the number and size of LDs during starvation, two-photon 3D imaging to compare the variations of LDs in various liver tissues, and STED super-resolution imaging to visualize the nanoscale LDs with the resolution of 65 nm. Overall, these imaging findings validate the effectiveness of the new strategy for improving the labeling selectivity.

β-Galactosidase- and Photo-Activatable Fluorescent Probes for Protein Labeling and Super-Resolution STED Microscopy in Living Cells.

We report on the synthesis of two fluorescent probes which can be activated by β-Galactosidase (β-Gal) enzymes and/or light. The probes contained 2-nitro-4-oxybenzyl and 3-nitro-4-oxybenzyl fragments, with β-Gal residues linked to C-4. We performed the enzymatic and photoactivation of the probes in a cuvette and compared them, prior to the labeling of Vimentin-Halo fusion protein in live cells with overexpressed β-galactosidase. The dye fluorescence afforded the observation of enzyme activity by means of confocal and super-resolution optical microscopy based on stimulated emission depletion (STED). The tracing of enzymatic activity with the retention of activated fluorescent products inside cells was combined with super-resolution imaging as a tool for use in biomedicine and life science.

Surface-Functionalized Halo-Tag Gold Nanoprobes for Live-Cell Long-Term Super-Resolution Imaging of Endoplasmic Reticulum Dynamics.

Super-resolution fluorescence microscopy has emerged as a powerful tool for studying endoplasmic reticulum (ER) dynamics in living cells. However, the lack of high-brightness, high-photostability, and stable labeling probes makes long-term super-resolution imaging of the ER still challenging. Herein, we reported a surface-functionalized Halo-tag gold nanofluorescent probe (GNP-Atto565-fR8-CA) that exhibits excellent brightness, photostability, and biocompatibility. GNP-Atto565-fR8-CA can simultaneously load multiple Atto565 dye molecules, significantly improving its brightness. Modifying the cell-penetrating peptide fR8 enables GNP-Atto565-fR8-CA to be efficiently delivered into the cytoplasm, overcoming the challenge of their easy entrapment in vesicles. Fluorescent labeling of ER proteins via Halo tags enables high specificity and stable labeling of GNP-Atto565-fR8-CA to the ER. The SIM super-resolution imaging results showed that GNP-Atto565-fR8-CA can track and observe the long-term dynamic process of the ER, and can also be used for long-term super-resolution imaging of the dynamic interactions between the ER and other organelles. This work offers a practical tool to study live-cell ER ultrastructure and dynamics.

A Water-Soluble Multifunctional Probe for Colorimetric Copper Sensing, Lysosome Labelling and Live-Cell Imaging.

We report a water-soluble fluorescence and colorimetric copper probe (LysoBC1); this system can also serve for lysosome labeling and for the dynamic tracking of Cu2+ in living cells. The sensing mechanism takes advantage of the synergic action by the following three components: i) a lysosome targeting unit, ii) the spirolactam ring-opening for the selective copper chelation and iii) the metal-mediated hydrolysis of the rhodamine moiety for fluorescence enhancement. In aqueous environment the molecule acts as a fluorescent reversible pH sensor and as colorimetric probe for Cu2+ at physiological pH; the hydrolysis of the copper targeting unit resulted in a 50-fold increase of the fluorescence intensity. Most importantly, in vitro cell analyses in undifferentiated (SH SY5Y) and differentiated (d-SH SY5Y) neuroblastoma cells, LysoBC1 is able to selectively accumulate into lysosome while the copper binding ability allowed us to monitor intracellular copper accumulation into lysosome.

Photoactivatable mRNA 5' Cap Analogs for RNA-Protein Crosslinking.

Chemical modification of messenger RNA (mRNA) has paved the way for advancing mRNA-based therapeutics. The intricate process of mRNA translation in eukaryotes is orchestrated by numerous proteins involved in complex interaction networks. Many of them bind specifically to a unique structure at the mRNA 5'-end, called 5'-cap. Depending on the 5'-terminal sequence and its methylation pattern, different proteins may be involved in the translation initiation and regulation, but a deeper understanding of these mechanisms requires specialized molecular tools to identify natural binders of mRNA 5'-end variants. Here, a series of 8 new synthetic 5'-cap analogs that allow the preparation of RNA molecules with photoreactive tags using a standard in vitro transcription reaction are reported. Two photoreactive tags and four different modification sites are selected to minimize potential interference with cap-protein contacts and to provide complementary properties regarding crosslinking chemistry and molecular interactions. The tailored modification strategy allows for the generation of specific crosslinks with model cap-binding proteins, such as eIF4E and Dcp2. The usefulness of the photoreactive cap analogs is also demonstrated for identifying the cap-binding subunit in a multi-protein complex, which makes them perfect candidates for further development of photoaffinity labeling probes to study more complex mRNA-related processes.

N-Acyl-N-Alkyl Sulfonamide Probes for Ligand-Directed Covalent Labeling of GPCRs: The Adenosine A2B Receptor as Case Study.

Small molecular tool compounds play an essential role in the study of G protein-coupled receptors (GPCRs). However, tool compounds most often occupy the orthosteric binding site, hampering the study of GPCRs upon ligand binding. To overcome this problem, ligand-directed labeling techniques have been developed that leave a reporter group covalently bound to the GPCR, while allowing subsequent orthosteric ligands to bind. In this work, we applied such a labeling strategy to the adenosine A2B receptor (A2BAR). We have synthetically implemented the recently reported N-acyl-N-alkyl sulfonamide (NASA) warhead into a previously developed ligand and show that the binding of the A2BAR is not restricted by NASA incorporation. Furthermore, we have investigated ligand-directed labeling of the A2BAR using SDS-PAGE, flow cytometric, and mass spectrometry techniques. We have found one of the synthesized probes to specifically label the A2BAR, although detection was hindered by nonspecific protein labeling most likely due to the intrinsic reactivity of the NASA warhead. Altogether, this work aids the future development of ligand-directed probes for the detection of GPCRs.