Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

One-Step Azolation Strategy for Site- and Chemo-Selective Labeling of Proteins with Mass-Sensitive Probes.

ABSTRACT: The chemical modification of proteins in a site-selective manner leads to many advances in various scientific fields. The major challenges with conventional N-terminal bioconjugation techniques are the lack of universal sequence compatibility and poor mass-detection sensitivity of the resulting bioconjugates. This approach efficiently analyzes proteolytic fragments and native proteins in a complex mixture. Multiple chemical steps are usually required for the site-selective synthesis of bioconjugates with enhanced mass-detection sensitivity. We present a single-step, versatile strategy for the selective modification of protein N-termini with mass boosters. The chemical tag enhances the peptide detection by multiple orders thus leading to the unambiguous analysis of the resulting bioconjugates. We demonstrate that tagging proteolytic fragments with mass sensitivity probes in a complex mixture improves the detection of resulting bioconjugates.

SUBMITTER: Tang KC

PROVIDER: S-EPMC10111340 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

One-Step Azolation Strategy for Site- and Chemo-Selective Labeling of Proteins with Mass-Sensitive Probes.

Tang Kuei C KC Raj Monika M

Angewandte Chemie (International ed. in English) 20201130 4

The chemical modification of proteins in a site-selective manner leads to many advances in various scientific fields. The major challenges with conventional N-terminal bioconjugation techniques are the lack of universal sequence compatibility and poor mass-detection sensitivity of the resulting bioconjugates. This approach efficiently analyzes proteolytic fragments and native proteins in a complex mixture. Multiple chemical steps are usually required for the site-selective synthesis of bioconjug ...[more]

PMID: 33047860

Similar Datasets

One-Step Selective Exoenzymatic Labeling (SEEL) Strategy for the Biotinylation and Identification of Glycoproteins of Living Cells.

Project description:Technologies that can visualize, capture, and identify subsets of biomolecules that are not encoded by the genome in the context of healthy and diseased cells will offer unique opportunities to uncover the molecular mechanism of a multitude of physiological and disease processes. We describe here a chemical reporter strategy for labeling of cell surface glycoconjugates that takes advantage of recombinant glycosyltransferases and a corresponding sugar nucleotide functionalized by biotin. The exceptional efficiency of this method, termed one-step selective exoenzymatic labeling, or SEEL, greatly improved the ability to enrich and identify large numbers of tagged glycoproteins by LC-MS/MS. We further demonstrated that this labeling method resulted in far superior enrichment and detection of glycoproteins at the plasma membrane compared to a sulfo-NHS-activated biotinylation or two-step SEEL. This new methodology will make it possible to profile cell surface glycoproteomes with unprecedented sensitivity in the context of physiological and disease states.

| S-EPMC5067740 | biostudies-literature

A One-Step Chemoenzymatic Labeling Strategy for Probing Sialylated Thomsen-Friedenreich Antigen.

Project description:Abnormal expression of sialylated Thomsen-Friedenreich antigen (Neu5Acα2-3Galβ1-3GalNAcα-O-Ser/Thr, sialyl-T) has a strong relationship with various types of human cancers and many other diseases. However, the size and structural complexity, and relatively lower abundance of sialyl-T have posed a significant challenge to its detection. Therefore, details about the role of sialyl-T in a variety of physiological and pathological processes are still poorly understood. Here, a one-step chemoenzymatic labeling strategy to probe sialyl-T is described. This approach enables the sensitive, selective, and rapid detection of sialyl-T, and global profiling and identification of unknown sialyl-T-attached glycoproteins, which are potential therapeutic targets or biomarkers. The use of one-step labeling strategy not only has a higher sensitivity than a typical two-step reporter strategy but also avoids undergoing an additional chemical reaction step to introduce a reporter group after the labeling reaction, making it particularly useful for detecting low-abundance glycan epitopes on living cells.

| S-EPMC5920613 | biostudies-literature

One-Step 18F-Labeling and Preclinical Evaluation of Prostate-Specific Membrane Antigen Trifluoroborate Probes for Cancer Imaging.

Project description:After the identification of the high-affinity glutamate-ureido scaffold, the design of several potent 18F- and 68Ga-labeled tracers has allowed spectacular progress in imaging recurrent prostate cancer by targeting the prostate-specific membrane antigen (PSMA). We evaluated a series of PSMA-targeting probes that are 18F-labeled in a single step for PET imaging of prostate cancer. Methods: We prepared 8 trifluoroborate constructs for prostate cancer imaging, to study the influence of the linker and the trifluoroborate prosthetic on pharmacokinetics and image quality. After 1-step labeling by 19F-18F isotopic exchange, the radiotracers were injected in mice bearing LNCaP xenografts, with or without blocking controls, to assess specific uptake. PET/CT images and biodistribution data were acquired at 1 h after injection and compared with 18F-DCFPyL on the same mouse strain and tumor model. Results: All tracers exhibited nanomolar affinities, were labeled in good radiochemical yields at high molar activities, and exhibited high tumor uptake in LNCaP xenografts with clearance from nontarget organs. Most derivatives with a naphthylalanine linker showed significant gastrointestinal excretion. A radiotracer incorporating this linker with a dual trifluoroborate-glutamate labeling moiety showed high tumor uptake, low background activity, and no liver or gastrointestinal track accumulation. Conclusion: PSMA-targeting probes with trifluoroborate prosthetic groups represent promising candidates for prostate cancer imaging because of facile labeling while affording high tumor uptake values and contrast ratios that are similar to those obtained with 18F-DCFPyL.

| S-EPMC6681697 | biostudies-literature

Broad substrate tolerance of tubulin tyrosine ligase enables one-step site-specific enzymatic protein labeling.

Project description:The broad substrate tolerance of tubulin tyrosine ligase is the basic rationale behind its wide applicability for chemoenzymatic protein functionalization. In this context, we report that the wild-type enzyme enables ligation of various unnatural amino acids that are substantially bigger than and structurally unrelated to the natural substrate, tyrosine, without the need for extensive protein engineering. This unusual substrate flexibility is due to the fact that the enzyme's catalytic pocket forms an extended cavity during ligation, as confirmed by docking experiments and all-atom molecular dynamics simulations. This feature enabled one-step C-terminal biotinylation and fluorescent coumarin labeling of various functional proteins as demonstrated with ubiquitin, an antigen binding nanobody, and the apoptosis marker Annexin V. Its broad substrate tolerance establishes tubulin tyrosine ligase as a powerful tool for in vitro enzyme-mediated protein modification with single functional amino acids in a specific structural context.

| S-EPMC5418632 | biostudies-literature

Thiol-selective fluorogenic probes for labeling and release.

Project description:Thiol alkylation is a powerful technique for the labeling of proteins. We report a new class of highly reactive, selective, and fluorogenic probes for thiols in aqueous solution at neutral pH, based on the 7-oxanorbornadiene (OND) framework. The maleate moiety in 7-oxabicyclo[2.2.1]hept-2,5-diene-2,3-dicarboxylic acid esters serves as both a tunable electrophile and an intramolecular quencher of an attached dansyl fluorophore. Thiols have been found to add with high rates (second-order rate constants of 40-200 M(-1) s(-1)) to give adducts that exhibit enhancements of fluorescence intensity up to 180-fold. The resulting adducts are also versatile with respect to cleavage (release) reactions by two mechanisms. First, retro-Diels-Alder fragmentation occurs with half-lives from days to weeks at room temperature, and an epoxide derivative is also reported that is incapable of cycloreversion cleavage. Second, monoamide OND derivatives undergo rapid closure to succinimides upon thiol addition, providing a thiol-triggered mechanism for immediate alcohol release. Peptides and proteins containing free thiol groups were labeled with OND electrophiles with high chemoselectivity. Since the system is so easily assembled from readily accessible modules, various functional groups can be added to OND linkers to allow the attachment of other molecules of interest.

| S-EPMC3166955 | biostudies-literature

Sensitive luciferin derived probes for selective carboxypeptidase activity.

Project description:Highly selective luminescent probes, QLUC-TYR and LUC-GLU, for detection of carboxypeptidase activity were synthesized. Caged substrates were first cleaved by corresponding carboxypeptidases, and then they were activated by luciferase to emit light. Enzymatic activities of biologically important carboxypeptidases can be determined using this technology.

| S-EPMC3129288 | biostudies-literature

A novel strategy for site selective spin-labeling to investigate bioactive entities by DNP and EPR spectroscopy.

Project description:A novel specific spin-labeling strategy for bioactive molecules is presented for eptifibatide (integrilin) an antiplatelet aggregation inhibitor, which derives from the venom of certain rattlesnakes. By specifically labeling the disulfide bridge this molecule becomes accessible for analytical techniques such as Electron Paramagnetic Resonance (EPR) and solid state Dynamic Nuclear Polarization (DNP). The necessary spin-label was synthesized and inserted into the disulfide bridge of eptifibatide via reductive followed by insertion by a double Michael addition under physiological conditions. This procedure is universally applicable for disulfide containing biomolecules and is expected to preserve their tertiary structure with minimal change due to the small size of the label and restoring of the previous disulfide connection. HPLC and MS analysis show the successful introduction of the spin label and EPR spectroscopy confirms its activity. DNP-enhanced solid state NMR experiments show signal enhancement factors of up to 19 in 13C CP MAS experiments which corresponds to time saving factors of up to 361. This clearly shows the high potential of our new spin labeling strategy for the introduction of site selective radical spin labels into biomolecules and biosolids without compromising its conformational integrity for structural investigations employing solid-state DNP or advanced EPR techniques.

| S-EPMC8249612 | biostudies-literature

Highly stable and sensitive fluorescent probes (LysoProbes) for lysosomal labeling and tracking.

Project description:We report the design, synthesis and application of several new fluorescent probes (LysoProbes I-VI) that facilitate lysosomal pH monitoring and characterization of lysosome-dependent apoptosis. LysoProbes are superior to commercially available lysosome markers since the fluorescent signals are both stable and highly selective, and they will aid in characterization of lysosome morphology and trafficking. We predict that labeling of cancer cells and solid tumor tissues with LysoProbes will provide an important new tool for monitoring the role of lysosome trafficking in cancer invasion and metastasis.

| S-EPMC4341211 | biostudies-literature

Selective Labeling of Proteins on Living Cell Membranes Using Fluorescent Nanodiamond Probes.

Project description:The impeccable photostability of fluorescent nanodiamonds (FNDs) is an ideal property for use in fluorescence imaging of proteins in living cells. However, such an application requires highly specific labeling of the target proteins with FNDs. Furthermore, the surface of unmodified FNDs tends to adsorb biomolecules nonspecifically, which hinders the reliable targeting of proteins with FNDs. Here, we combined hyperbranched polyglycerol modification of FNDs with the β-lactamase-tag system to develop a strategy for selective imaging of the protein of interest in cells. The combination of these techniques enabled site-specific labeling of Interleukin-18 receptor alpha chain, a membrane receptor, with FNDs, which eventually enabled tracking of the diffusion trajectory of FND-labeled proteins on the membrane surface.

| S-EPMC5302567 | biostudies-literature

Building addressable libraries: a site-selective click-reaction strategy for rapidly assembling mass spectrometry cleavable linkers.

Project description:A click-reaction was site-selectively carried out on 1000 and 12,000 microelectrode arrays and characterized using TOF-SIMS.

| S-EPMC3958968 | biostudies-literature