67 research outputs found
Direct Profiling the Post-Translational Modification Codes of a Single Protein Immobilized on a Surface Using Cu-free Click Chemistry
Combinatorial post-translational modifications (PTMs), which can serve as dynamic molecular barcodes, have been proposed to regulate distinct protein functions. However, studies of combinatorial PTMs on single protein molecules have been hindered by a lack of suitable analytical methods. Here, we describe erasable single-molecule blotting (eSiMBlot) for combinatorial PTM profiling. This assay is performed in a highly multiplexed manner and leverages the benefits of covalent protein immobilization, cyclic probing with different antibodies, and single molecule fluorescence imaging. Especially, facile and efficient covalent immobilization on a surface using Cu-free click chemistry permits multiple rounds (>10) of antibody erasing/reprobing without loss of antigenicity. Moreover, cumulative detection of coregistered multiple data sets for immobilized single-epitope molecules, such as HA peptide, can be used to increase the antibody detection rate. Finally, eSiMBlot enables direct visualization and quantitative profiling of combinatorial PTM codes at the single-molecule level, as we demonstrate by revealing the novel phospho-codes of ligand-induced epidermal growth factor receptor. Thus, eSiMBlot provides an unprecedentedly simple, rapid, and versatile platform for analyzing the vast number of combinatorial PTMs in biological pathways. Copyright © 2018 American Chemical Societ
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The Rate of Charge Tunneling through Self-Assembled Monolayers Is Insensitive to Many Functional Group Substitutions
Insensitivity: A series of molecules containing a common head group and body as well as structurally varied tail groups (-R) has been used in junctions with the structure Ag/S(CH2)4CONH(CH2)2R//Ga2O3/EGaIn to study the rates of charge transport by tunneling. Changing the structure of R over a range of common aliphatic, aromatic, and heteroaromatic organic groups was found to not significantly influence the rate of tunneling (see plots; the dashed lines represent calibration standards).Chemistry and Chemical Biolog
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Polymer-based mesh as supports for multi-layered 3D cell culture and assays
Three-dimensional (3D) culture systems can mimic certain aspects of the cellular microenvironment found in vivo, but generation, analysis and imaging of current model systems for 3D cellular constructs and tissues remain challenging. This work demonstrates a 3D culture system–Cells-in-Gels-in-Mesh (CiGiM)–that uses stacked sheets of polymer-based mesh to support cells embedded in gels to form tissue-like constructs; the stacked sheets can be disassembled by peeling the sheets apart to analyze cultured cells—layer-by-layer—within the construct. The mesh sheets leave openings large enough for light to pass through with minimal scattering, and thus allowing multiple options for analysis—(i) using straightforward analysis by optical light microscopy, (ii) by high-resolution analysis with fluorescence microscopy, or (iii) with a fluorescence gel scanner. The sheets can be patterned into separate zones with paraffin film-based decals, in order to conduct multiple experiments in parallel; the paraffin-based decal films also block lateral diffusion of oxygen effectively. CiGiM simplifies the generation and analysis of 3D culture without compromising throughput, and quality of the data collected: it is especially useful in experiments that require control of oxygen levels, and isolation of adjacent wells in a multi-zone format.Chemistry and Chemical Biolog
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A Bio-Inspired Swellable Microneedle Adhesive for Mechanical Interlocking with Tissue
Achieving significant adhesion to soft tissues while minimizing tissue damage poses a considerable clinical challenge. Chemical-based adhesives require tissue-specific reactive chemistry, typically inducing a significant inflammatory response. Staples are fraught with limitations including high-localized tissue stress and increased risk of infection, and nerve and blood vessel damage. Here, inspired by the endoparasite Pomphorhynchus laevis which swells its proboscis to attach to its host’s intestinal wall, we have developed a biphasic microneedle array that mechanically interlocks with tissue through swellable microneedle tips, achieving ~ 3.5 fold increase in adhesion strength compared to staples in skin graft fixation, and removal force of ~ 4.5 N/cm2 from intestinal mucosal tissue. Comprising a poly(styrene)-block-poly(acrylic acid) swellable tip and non-swellable polystyrene core, conical microneedles penetrate tissue with minimal insertion force and depth, yet high adhesion strength in their swollen state. Uniquely, this design provides universal soft tissue adhesion with minimal damage, less traumatic removal, reduced risk of infection and delivery of bioactive therapeutics
Bio-orthogonal Supramolecular Latching inside Live Animals and Its Application for in Vivo Cancer Imaging
© 2019 American Chemical Society.Here, we demonstrate a supramolecular latching tool for bio-orthogonal noncovalent anchoring of small synthetic molecules in live animal models using a fully synthetic high-affinity binding pair between cucurbit[7]uril (CB[7]) and adamantylammonium (AdA). This supramolecular latching system is small (∼1 kDa), ensuring efficient uptake into cells, tissues, and whole organisms. It is also chemically robust and resistant to enzymatic degradation and analogous to well-characterized biological systems in terms of noncovalent binding. Occurrence of fluorescence resonance energy transfer (FRET) between cyanine 3-CB[7] (Cy3-CB[7]) and boron-dipyrromethene 630/650X-AdA (BDP630/650-AdA) inside a live worm (Caenorhabditis elegans) indicates efficient in situ high-affinity association between AdA and CB[7] inside live animals. In addition, selective visualization of a cancer site of a live mouse upon supramolecular latching of cyanine 5-AdA (Cy5-AdA) on prelocalized CB[7]-conjugating antibody on the cancer site demonstrates the potential of this synthetic system for in vivo cancer imaging. These findings provide a fresh insight into the development of new chemical biology tools and medical therapeutic systems11sciescopu
Clinical Predictors of Incomplete ST-Segment Resolution in the Patients With Acute ST Segment Elevation Myocardial Infarction
Ultrastable artificial binding pairs as a supramolecular latching system: A next generation chemical tool for proteomics
In this Commentary, we discuss cucurbit[7]uril-based ultrastable artificial binding pairs as a supramolecular latching system and how we envision this becoming important tools in proteomics. The limitations of current proteomic techniques are described with an emphasis on the lack of tools to answer questions about the complex and dynamic nature of the proteome. Our thoughts as to how artificial ultrastable binding pairs may be able to address these questions are given especially when they are combined with existing methods. © 2017 American Chemical Society. © 2017 American Chemical Society113141sciescopu
Identification of mitochondrial proteins associated with cyanine 5 using a combination of affinity-based photocrosslinking with bio-orthogonal supramolecular latching
Visualization of mitochondria with cyanine dyes has been exploited to study the functions of mitochondria. However, proteins interacting with cyanine dyes have not been investigated clearly. Here, we report a chemical proteomics approach for the identification of mitochondrial proteins associated with cyanine dyes. This approach uses affinity-based photocrosslinking of Cy5-diazirine-alkyne (Cy5-DAyne) in combination with selective enrichment of target proteins by bio-orthogonal supramolecular latching based on unique host-guest interaction between cucurbit[7]uril (CB[7]) and its selected guest such as adamantylammonium (AdA). The enriched proteins then were analyzed using LC-MS/MS to provide a list of proteins closely associated with the Cy5 motif. This study suggests that the proteomic environment around the Cy5 motif is highly associated with energy production processes, including the respiratory chain, ATP synthesis, and fatty acid oxidization. Moreover, it demonstrates the potential of this chemical proteomic approach for the investigation of other organellar proteins using different selective dyes.11Nsciescopu
Guest-responsive, Non-proteolytic Harvest of a Cell-sheet using Controllable Host-guest Chemistry
A new non-proteolytic method to harvest a cell-sheet was demonstrated using controllable host-guest interactions which can be dissociated by treating a strong guest on demand. Fibroblast cells (NIH3T3) were grown to confluence on a 1,6-diammoniumhexane conjugated hyaluronic acid (DAH-HA) polymer which was anchored to a cucurbit[7]uril (CB[7]) surface using the host-guest interaction between DAH and CB[7]. Treating with a strong guest allowed the cultured cells to be detached from the surface as a free standing sheet. This approach demonstrated the great potential of controllable host-guest chemistry as a novel tool for non-proteolytic harvesting of cell-sheets useful for regenerative therapy. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinhei
Supramolecular hyper-branch polymerization using Host-guest interaction
Among supramolecular chemistry, host-guest interactions describe two molecules that interact based on non-covalent bonds. Non-covalent bonds are weaker than covalent bonds but are used to make large molecules such as proteins and nucleic acids that are important for biochemical processes. In this work, we designed AOCB[6]-PY, in which host and guest molecules are linked, to become a tunable hyperbranched polymer through host-guest interactions. Cucurbit [6] uril (CB [6]) is a non-toxic host material that does not fully interact with the guest material pyridine at room temperature, but at high temperatures the activity of the host and guest molecules increases, forming a hyperbranched polymer. Therefore, we developed reusable hyperbranched polymers using temperature-dependent host-guest interactions to achieve antimicrobial polymer coatings
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