Probe Labeling Research Articles

Molecular Mechanisms of Intracellular Delivery of Nanoparticles Monitored by an Enzyme-Induced Proximity Labeling

HighlightsNovel enzyme-induced proximity labeling technology in nanoparticles (nano-EPL).Nano-EPL enables dynamic molecular mapping of the intracellular delivery of nanoparticles in macrophages.Nano-EPL enables the elucidation of a comprehensive phagosome-centered timeline during the vesicular transportation of nanoparticles, revealing distinct organelle engagement and its differential impact on drug delivery efficiency.

Activity-based labelling of ammonia- and alkane-oxidizing microorganisms including ammonia-oxidizing archaea.

Recently, an activity-based labelling protocol for the in vivo detection of ammonia- and alkane-oxidizing bacteria became available. This functional tagging technique enabled targeted studies of these environmentally widespread functional groups, but it failed to capture ammonia-oxidizing archaea (AOA). Since their first discovery, AOA have emerged as key players within the biogeochemical nitrogen cycle, but our knowledge regarding their distribution and abundance in natural and engineered ecosystems is mainly derived from PCR-based and metagenomic studies. Furthermore, the archaeal ammonia monooxygenase is distinctly different from its bacterial counterparts and remains poorly understood. Here, we report on the development of an activity-based labelling protocol for the fluorescent detection of all ammonia- and alkane-oxidizing prokaryotes, including AOA. In this protocol, 1,5-hexadiyne is used as inhibitor of ammonia and alkane oxidation and as bifunctional enzyme probe for the fluorescent labelling of cells via the Cu(I)-catalyzed alkyne-azide cycloaddition reaction. Besides efficient activity-based labelling of ammonia- and alkane-oxidizing microorganisms, this method can also be employed in combination with deconvolution microscopy for determining the subcellular localization of their ammonia- and alkane-oxidizing enzyme systems. Labelling of these enzymes in diverse ammonia- and alkane-oxidizing microorganisms allowed their visualization on the cytoplasmic membranes, the intracytoplasmic membrane stacks of ammonia- and methane-oxidizing bacteria, and, fascinatingly, on vesicle-like structures in one AOA species. The development of this novel activity-based labelling method for ammonia- and alkane-oxidizers will be a valuable addition to the expanding molecular toolbox available for research of nitrifying and alkane-oxidizing microorganisms.

Bioisostere-conjugated fluorescent probes for live-cell protein imaging without non-specific organelle accumulation.

Specific labeling of proteins using membrane-permeable fluorescent probes is a powerful technique for bioimaging. Cationic fluorescent dyes with high fluorescence quantum yield, photostability, and water solubility provide highly useful scaffolds for protein-labeling probes. However, cationic probes generally show undesired accumulation in organelles, which causes a false-positive signal in localization analysis. Herein, we report a design strategy for probes that suppress undesired organelle accumulation using a bioisostere for intracellular protein imaging in living cells. Our design allows the protein labeling probes to possess both membrane permeability and suppress non-specific accumulation and has been shown to use several protein labeling systems, such as PYP-tag and Halo tag systems. We further developed a fluorogenic PYP-tag labeling probe for intracellular proteins and used it to visualize multiple localizations of target proteins in the intracellular system. Our strategy offers a versatile design for undesired accumulation-suppressed probes with cationic dye scaffolds and provides a valuable tool for intracellular protein imaging.

Dichlorotriazine-based multivalent probe for selective affinity labeling of carbohydrate-binding proteins.

A new series of multivalent gold nanoparticle probes bearing different electrophilic groups were synthesized and their affinity labeling reactivities were evaluated. The dichlorotriazine group was identified as a useful protein-reactive label, allowing selective capture of a target protein at nanomolar probe concentrations.

Mechanistic differences between linear vs. spirocyclic dialkyldiazirine probes for photoaffinity labeling.

Dialkyldiazirines have emerged as a photo-reactive group of choice for interactome mapping in live cell experiments. Upon irradiation, 'linear' dialkyldiazirines produce dialkylcarbenes which are susceptible to both intramolecular reactions and unimolecular elimination processes, as well as diazoalkanes, which also participate in intermolecular labeling. Cyclobutylidene has a nonclassical bonding structure and is stable enough to be captured in bimolecular reactions. Cyclobutanediazirines have more recently been studied as photoaffinity probes based on cyclobutylidene, but the mechanism, especially with respect to the role of putative diazo intermediates, was not fully understood. Here, we show that photolysis (365 nm) of cyclobutanediazirines can produce cyclobutylidene intermediates as evidenced by formation of their expected bimolecular and unimolecular products, including methylenecyclopropane derivatives. Unlike linear diazirines, cyclobutanediazirine photolysis in the presence of tetramethylethylene produces a [2 + 1] cycloaddition adduct. By contrast, linear diazirines produce diazo compounds upon low temperature photolysis in THF, whereas diazo compounds are not detected in similar photolyses of cyclobutanediazirines. Diazocyclobutane, prepared by independent synthesis, is labile, reactive toward water and capable of protein alkylation. The rate of diazocyclobutane decomposition is not affected by 365 nm light, suggesting that the photochemical conversion of diazocyclobutane to cyclobutylidene is not an important pathway. Finally, chemical proteomic studies revealed that a likely consequence of this primary conversion to a highly reactive carbene is a marked decrease in labeling by cyclobutanediazirine-based probes relative to linear diazirine counterparts both at the individual protein and proteome-wide levels. Collectively, these observations are consistent with a mechanistic picture for cyclobutanediazirine photolysis that involves carbene chemistry with minimal formation of diazo intermediates, and contrasts with the photolyses of linear diazirines where alkylation by diazo intermediates plays a more significant role.

Protein painting for structural and binding site analysis via intracellular lysine reactivity profiling with o-phthalaldehyde.

The three-dimensional structure and the molecular interaction of proteins determine their roles in many cellular processes. Chemical protein painting with protein mass spectrometry can identify changes in structural conformations and molecular interactions of proteins including their binding sites. Nevertheless, most current protein painting techniques identify protein targets and binding sites of drugs in vitro using a cell lysate or purified protein. Here, we tested 11 membrane-permeable lysine-reactive chemical probes for intracellular covalent labeling of endogenous proteins, which reveals ortho-phthalaldehyde (OPA) as the most reactive probe in the intracellular environment. An MS workflow and a new data analysis strategy termed RAPID (Reactive Amino acid Profiling by Inverse Detection) was developed to enhance detection sensitivity. RAPID with OPA successfully identified structural changes induced by the allosteric drug TEPP-46 on its target protein PKM2 and was applied to profile the conformation change of the proteome occurring in cells during thermal denaturation. The application of RAPID-OPA on cells treated with geldanamycin, selumetinib, and staurosporine successfully revealed their binding sites on target proteins. Thus, RAPID-OPA for cellular protein painting enables the identification of ligand-binding sites and detection of protein structural changes occurring in cells.

Design, synthesis and modelling of photoreactive chemical probes for investigating target engagement of plasmepsin IX and X in Plasmodium falciparum.

The emergence of Plasmodium parasite resistance to current front-line antimalarial treatments poses a serious threat to global malaria control and highlights the necessity for the development of therapeutics with novel targets and mechanisms of action. Plasmepsins IX and X (PMIX/PMX) have been recognised as highly promising targets in Plasmodium due to their contribution to parasite's pathogenicity. Recent research has demonstrated that dual PMIX/PMX inhibition results in the impairment of multiple parasite's life cycle stages, which is an important feature in drug resistance prevention. Herein we report novel hydroxyethylamine photoaffinity labelling (PAL) probes, designed for PMIX/PMX target engagement and proteomics experiments in Plasmodium parasites. The prepared probes have both a photoreactive group (diazirine or benzophenone) for covalent attachment to target proteins, and a terminal alkyne handle allowing their use in bioorthogonal ligation. One of the synthesised benzophenone probes was shown to be highly promising as demonstrated by its outstanding antimalarial potency (IC50 = 15 nM versus D10 P. falciparum) and its inhibitory effect against PfPMX in an enzymatic assay. Molecular docking and molecular dynamics studies show that the inclusion of the benzophenone and alkyne handle does not alter the binding mode compared to the parent compound. The photoaffinity probe can be used in future chemical proteomics studies to allow hydroxyethylamine drug scaffold target identification and validation in Plasmodium. We expect our findings to act as a tool for future investigations on PMIX/PMX inhibition in antimalarial drug discovery.

Gold brocade coated CoFe PBA with enhanced peroxidase-like activity for a chemiluminescent imaging immunoassay.

Traditional chemiluminescence (CL) imaging immunoassays usually rely on natural enzymes as catalytic probes, which has hampered their extensive application due to the susceptibility to inactivation of natural enzymes. In response, a gold brocade coated CoFe Prussian blue analogue (CoFe PBA@Au brocade) with enhanced peroxidase-like activity was synthesized and utilized as a powerful label probe for constructing a highly sensitive CL imaging immunosensor targeting disease biomarkers with excellent performance. This research offers a universal strategy for enhancing the sensitivity of CL imaging immunoassays and further expands the application of PBA nanozymes.

Covalent PROTAC design method based on a sulfonyl pyridone probe.

Covalent proteolysis-targeting chimeras (PROTACs) offer enhanced selectivity, prolonged action, and increased efficacy against challenging target proteins. The conventional approach relies on covalent ligands, but our study presents an innovative method employing an N-sulfonyl pyridone warhead to selectively target tyrosine (Tyr) residues. The von Hippel-Lindau (VHL) moiety is transferred from the warhead to the exposed Tyr, allowing us to design a STING degrader (DC50 0.53 μM, Dmax 56.65%). This approach showcases the potential of nucleophilic amino acid labeling probes, particularly for proteins lacking easily accessible cysteine residues, opening new possibilities for covalent PROTAC design and targeted protein degradation therapies.

12-Plex Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tag-Enabled High-Throughput Quantitative Glycomics.

Glycans, which are ubiquitously distributed on most proteins and cell surfaces, are a class of important biomolecules playing crucial roles in various biological processes such as molecular recognition and cellular communication. Modern mass spectrometry (MS) coupled with novel chemical probe labeling strategies has greatly advanced analysis of glycans. However, the requirement of high-throughput and robust quantitative analysis still calls for the development of more advanced tools. Recently, we devised isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags for 4-plex N-glycan analysis. To further improve the throughput, we utilized the mass-defect strategy and expanded the multiplexing capacity to 12 channels without changing the chemical structure of the SUGAR tag, achieving a threefold enhancement in throughput compared with the original design and managing to perform high-throughput N-glycan analysis in a single LC-MS/MS injection. Herein, we present detailed methods for the synthesis of 12-plex SUGAR isobaric tags, the procedure to release and label the N-glycans from proteins, and the analysis by high-resolution LC-MS/MS, as well as data processing to achieve multiplexed quantitative glycomics.

Time-resolved fluorescence immunoassay based on glucose oxidase-encapsulated metal-organic framework for amplified detection of foodborne pathogen

BackgroundFluorescence immunoassays are commonly employed for the detection of pathogenic bacteria as a means of ensuring food safety and preserving public health. However, the challenges such as poor photostability and background interference have limited their sensitivity and accuracy. The emergence of metal-organic frameworks (MOFs) as a label probe offers a promising solution for advancing fluorescence immunoassays owing to their tunable nature. Nonetheless, the low fluorescence efficiency of MOFs and the potential risk of dye leakage pose obstacles to achieving high detection sensitivity. Therefore, there exists a pressing need to fully utilize the potential of MOF composites in fluorescence immunoassays. ResultsWe explored the potential of glucose oxidase-encapsulated zeolitic imidazole framework-90 (GOx@ZIF-90) as a label probe to construct a time-resolved fluorescence immunoassay with amplified detection signal. This immunoassay involved functionalizing Fe3O4 nanoparticle with porcine antibody to specifically capture and separate the target bacteria, Staphylococcus aureus (S. aureus). The captured S. aureus was then bound by GOx@ZIF-90 modified with vancomycin, resulting in a fluorescence response in the europium tetracycline (EuTc). The encapsulation of GOx in ZIF-90 provided a confinement effect that significantly enhanced the catalytic activity and stability of GOx. This led to a highly efficient conversion of glucose to H2O2, amplifying the fluorescence signal of EuTc. The immunoassay demonstrated a high sensitivity in detecting S. aureus, with a detection limit of 2 CFU/mL. We also obtained satisfactory results in milk samples. Attractively, the time-resolved detection mode of EuTc allowed the immunoassay to eliminate background fluorescence and enhance accuracy. SignificanceThis study not only presented a new method for detecting foodborne pathogens but also highlighted the potential of enzyme-encapsulated MOF composites as label probes in immunoassays, providing valuable insights for the design and fabrication of MOF composites for various applications.

Carrier-free, pure nanodrugs based on dasatinib and 5-Fluorouracil show aggregation-induced emission and enhanced antitumor efficacy

Therapies based on small molecule targeted inhibitors usually suffer from several limitations such as poor water solubility, low oral bioavailability, rapid metabolism in vivo, and dose-limiting toxicity. The aim of this paper is to prepare a carrier-free nanodrug self-delivery system by self-assembly of dasatinib (DAS) and 5-Fluorouracil (Fu). By adjusting the ratio of DAS to Fu (10:1, 5:1, 2:1, 1:1), a series of nanoparticles (DAS/F NPs) with different particle sizes can be obtained without additional chemical modifications or carrier materials. More importantly, during self-assembly, DAS/F NPs produce aggregation-induced emission (AIE) effect. This makes DAS/F NPs suitable for bioimaging and tracking cell trafficking without any fluorescent probe labeling. DAS/F NPs (2:1) with a diameter around 150 nm was selected for the next antitumor study. The cellular uptake and cytotoxicity of DAS/F NPs were measured by EMT6 and MCF-7 cells as well as the corresponding multicellular spheroids. Finally, in vivo antitumor against EMT6 tumor-bearing mice, immunohistochemical studies and hemolysis assays demonstrated DAS/F NPs possess significant synergistic antitumor effects and excellent biocompatibility.

Development of Mitochondria‐Targeting Photosensitizers via Topoisomerase I Inhibition

AbstractPhotodynamic therapy (PDT) is an important oncologic intervention option for combating drug resistance of current chemotherapy in cancer. To improve spatiotemporal control, the development of subcellular organelle‐targeting photosensitizers is critical due to the diffusion limits of short‐lived ROS. Mitochondrial topoisomerase 1 (TOP1MT), exclusively localized in mitochondria, is emerging as a promising target for anticancer treatment. In this work, we designed and synthesized mitochondria‐targeting photosensitizers via Top1 inhibition activity of IQ6 and visualized the covalent adducts between the engaged proteins and the synthesized photoaffinity labeling probes (IQ‐Ps). Positively charged derivatives (IQ‐Ss) were also synthesized and their photo‐induced cytotoxicity was assessed to select IQ‐S1. Based on the cellular accumulation of IQ‐S1 in mitochondria and the enhanced PDT activity, we demonstrated that IQ‐S1 functions as a photosensitizer with sequential targeting of mitochondria and nucleus depending on irradiation times. Our findings suggest that the translocation of IQ‐S1 is related to the damage to mitochondrial Top1. The synthesized IQ‐S1 may contribute to the identification and tracking of cellular target proteins along with the PDT progression.

Small Plasma Membrane-Targeted Fluorescent Dye for Long-Time Imaging and Protein Degradation Analyses.

Plasma membrane (PM)-targeted fluorescent dyes have become an important tool to visualize morphological and dynamic changes in the cell membrane. However, most of these PM dyes are either too large and thus might potentially perturb the membrane and affect its functions or exhibit a short retention time on the cell membrane. The rapid internalization problem is particularly severe for PM dyes based on cationic and neutral hydrophobic fluorescent dyes, which can be easily transported into the cells by transmembrane potential and passive diffusion mechanisms. In this paper, we report a small but highly specific PM fluorescent dye, PM-1, which exhibits a very long retention time on the plasma membrane with a half-life of approximately 15 h. For biological applications, we demonstrated that PM-1 can be used in combination with protein labeling probes to study ectodomain shedding and endocytosis processes of cell surface proteins and successfully demonstrated that native transmembrane human carbonic anhydrase IX (hCAIX) is degraded via the ectodomain shedding mechanism. In contrast, hCAIX undergoes endocytic degradation in the presence of sheddase inhibitors. We believe that PM-1 can be a versatile tool to provide detailed insights into the dynamic processes of the cell surface proteins.

New-generation technologies for spatial tissue analysis, indispensable tools for deciphering intratumor heterogeneity in the development of antibody-drug conjugates and radio-immunoconjugates for cancer treatment.

Technologies allowing in situ tissue molecular analysis of the "high-plex" type (>20 molecules per tissue section) are the 21st century inventions that are revolutionizing our knowledge of the biology of malignant tumors and many benign alterations. These technologies are based on specific probe labeling systems for the detection of tissue components [proteins, messenger RNA (mRNA)], as well as on detailed image analysis, combined with computational tools. We are synthetically presenting technologies based on image analysis, such as multiplex immunofluorescence (mIF), imaging mass cytometry (IMC), and multiplexed ion beam imaging (MIBI), as well as the ones not based on image analysis, such as multiplex in situ hybridizations (ISHs) using various principles. All of them are supported by powerful software which enable both tissue segmentation and data analysis. In the context of cancer treatment personalization, these technologies can reveal areas of tumor tissue and/or cellular subpopulations that are responsible for good or bad responses to anticancer drugs. Thus, they represent an unprecedented aid in the exploration of intratumor heterogeneity (ITH), which has already been shown to be one of the main reasons for the therapeutic failure of targeted anticancer treatments. The arrival of antibody-drug conjugates (ADCs) and radio-immunoconjugates (RICs) in the therapeutic arsenal in oncology imposes a deep exploration of molecular ITH, where technologies of spatial tissue analysis reveal an emerging category of biomarkers-spatial biomarkers.

Novel Dumbbell-like CeVO4 Carrier-Based Immunochromatographic Assay for Highly Sensitive T-2 Toxin Detection in Food Samples.

Improving the sensitivity of immunochromatographic assays (ICAs) lies in the signal strength and probe activity of the labeled tracers, and the color properties and structure of the labeled tracers are key factors affecting the biological activity. In this study, cerium vanadate (CeVO4) of different sizes and shapes (230, 1058, and 710 nm) was synthesized to investigate its impact on the performance of ICA for T-2 detection. The prepared CeVO4 possessed outstanding stability, a large specific surface area, superior biocompatibility, and high compatibility with T-2 mAb (affinity constant was 3.14 × 108 M-1). As labeling probes for competitive ICA, the results showed that 1058 nm of CeVO4 as labels exhibited the best detection performance, with a limit of detection (LOD) of 0.079 ng/mL, which was substantially 19-fold less than the average of gold nanoparticle ICA. Additionally, CeVO4-ICA was effectively used to detect T-2 toxin, and the recovery rate for spiking corn and oatmeal samples was determined to be 81.27-115.44% (relative standard deviation <9.16%). The above information demonstrates the efficiency and applicability of CeVO4-ICA as a technique for quick and thorough identification of T-2 toxin residues in food.

Upconverting Nanoparticle-Based Photoactive Probes for Highly Efficient Labeling and Isolation of Target Proteins.

Photoaffinity labeling (PAL) has blossomed into a powerful and versatile tool for capture and identification of biomolecular targets. However, low labeling efficiency for specific targets such as lectins, the tedious process for protein purification, inevitable cellular photodamage, and less tissue penetration of UV light are significant challenges. Herein, we reported a near-infrared (NIR) light-driven photoaffinity labeling approach using upconverting nanoparticle (UCNP)-based photoactive probes, which were constructed by assembling photoactive groups and ligands onto NaYF4:Yb,Tm nanoparticles. The novel probes were easily prepared and functionalized, and the labeled proteins can be isolated and purified through simple centrifugation and washing. The advantages of this approach were demonstrated by labeling and isolation of peanut agglutinin (PNA), asialoglycoprotein receptor (ASGPR), and human carbonic anhydrase II (hCAII) from mixed proteins or cell lysates with good selectivity and efficiency, especially for PNA and ASGPR, two lectins that showed low binding affinity to their ligands. More importantly, successful labeling of PNA through pork tissues and ASGPR in mice strongly proved the good tissue penetrating capacity of NIR light and the application potential of UCNP-based photoactive probes for protein labeling in vivo. Biosafety of this approach was experimentally validated by enzyme, cell, and animal work, and we demonstrated that NIR light caused minimal photodamage to enzyme activity compared to UV light, and the UCNP-based photoactive probe presents good biosafety both in vitro and in vivo. We believe that this novel PAL approach will provide a promising tool for study of ligand-protein interactions and identification of biomolecular targets.

Highly sensitive lateral flow immunoassays based on Ag@Au nanoflowers with marine medaka (Oryzias melastigm) vitellogenin as a target analyte

In order to improve the sensitivity of lateral flow immunoassays (LFIAs) for the detection of piscine vitellogenin (Vtg), a well-established biomarker for environmental estrogens, Au coated Ag nanoflowers (Ag@Au NFs) were used as labeling probes to develop a LFIA for marine medaka Vtg. The synthesized Ag@Au NFs with good monodispersity had an average diameter of 44.1 nm and absorbance peak of 524 nm. When the concentration of goat anti-mouse IgG and anti-Vtg polyclonal antibody (anti-Vtg PAbs) were 1.3 and 0.4 mg/mL, respectively, the detection range of the LFIA was 0.19–25 ng/mL, and the visual detection limit was 0.1 ng/mL, which was approximately 80 times lower than that of LFIAs based on other nanoparticles (Au NPs, Ag NPs, Au NFs, and FM). After evaluation of its specificity and robustness, the usefulness of Ag@Au NFs labeled LFIA was validated by measuring Vtg induction in the plasma of marine medaka exposed to bisphenol A, a weak estrogenic chemical. This highly sensitive lateral flow immunoassay could detect Vtg biomarker within 15 min without the need of expensive and complicated instruments, and thus offered an ultrasensitive and robust on-site detection method for estrogenic activity in field environment.

Water–soluble and polarity–sensitive near–infrared fluorescent probe for long–time specific cancer cell membranes imaging and C. Elegans label

A novel D–π–A structure and near–infrared fluorescent probe (DCITT) with high polarity sensitivity and membrane targeting was reported. The fluorescent spectra of DCITT were polarity dependent and Stokes shift was greater than 300 nm. Due to its high fluorescence quantum yield, low cytotoxicity and photostability, DCITT could be used as a labeling probe in multicellular organisms. In particular, DCITT effectively distinguished tumor cells from normal cells because it could specifically light up the cancer cells membrane based on strong red fluorescence for a long time. On this basis, a polar–sensitive cell membrane probe is developed to differentiate tumor cells from normal cells, which provides an idea and method for the early diagnosis of tumor at cellular level.

Protocols for Efficient Chemical Synthesis of N2 -Modified Guanosine Phosphate Derivatives: Versatile Probes for mRNA Labeling.

A facile, reliable, and efficient method for the synthesis of N2 -modified guanosine nucleotides such as N2 -[benzyl-N-(propyl)carbamate]-guanosine-5'-O-monophosphate, N2 -[benzyl-N-(propyl)carbamate]-guanosine-5'-O-diphosphate, N2 -[benzyl-N-(propyl)carbamate]-guanosine-5'-O-triphosphate, and N2 -[benzyl-N-(propyl)carbamate]-N7 -methyl-guanosine-5'-O-diphosphate, starting from the corresponding nucleotide is described. The general process entails condensation between the exocyclic amine of guanosine nucleotide and 3-[(benzyloxycarbonyl)amino]propionaldehyde in aqueous methanol, followed by reduction using sodium cyanoborohydride to furnish the corresponding N2 -modified guanosine nucleotide in moderate yield with high purity (>99.5%). © 2023 Wiley Periodicals LLC. Basic Protocol: Synthesis of N2 -modified guanosine derivatives.