An engineered protein tag for multiprotein labeling in living cells

The visualization of complex cellular processes involving multiple proteins requires the use of spectroscopically distinguishable fluorescent reporters. We have previously introduced the SNAP-tag as a general tool for the specific labeling of SNAP-tag fusion proteins in living cells. The SNAP-tag is derived from the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) and can be covalently labeled in living cells using O6-benzylguanine derivatives bearing a chemical probe. Here we report the generation of an AGT-based tag, named CLIP-tag, which reacts specifically with O2-benzylcytosine derivatives. Because SNAP-tag and CLIP-tag possess orthogonal substrate specificities, SNAP and CLIP fusion proteins can be labeled simultaneously and specifically with different molecular probes in living cells. We furthermore show simultaneous pulse-chase experiments to visualize different generations of two different proteins in one sample.     

Affinity-guided protein conjugation: the trilogy of covalent protein labeling,assembly and inhibition

Specific and dynamic biological interactions pave the blueprint of signal networks in cell. For example, a great variety of specific protein-ligand interactions define how intracellular signals flow. Taking advantage of the specificity of these interactions, we postulate an “affinity-guided covalent conjugation” strategy to lock binding ligands through covalent reactions between the ligand and the receptor protein. The presence of a nucleophile close to the ligand binding site of a protein is sine qua none of this reaction. Specific noncovalent interaction of a ligand derivative (which contains an electrophile at a designed position) to the ligand binding site of the protein brings the electrophile to the close proximity of the nucleophile. Subsequently, a conjugation reaction spontaneously takes place between the nucleophile and the electrophile, and leads to an intermolecular covalent linkage. This strategy was first showcased in coiled coil peptides which include a cysteine mutation at a selected position. The short peptide sequence was used for covalent labeling of cell surface receptors. The same strategy was then used to guide the design of a set of protein Lego bricks for covalent assembly of protein complexes of unnatural geometry. We finally made “reactive peptides” for natural adaptor proteins that play significant roles in signal transduction. The peptides were designed to react with a single domain of the multidomain adaptor protein, delivered into the cytosol of neurons, and re-directed the intracellular signal of neuronal migration. The trilogy of protein labeling, assembly, and inhibition of intracellular signals, all through a specific covalent bond, fully demonstrated the generality and versatility of “affinity-guided covalent conjugation” in various applications.     

Routes to covalent catalysis by reactive selection for nascent protein nucleophiles

Reactivity-based selection strategies have been used to enrich combinatorial libraries for encoded biocatalysts having revised substrate specificity or altered catalytic activity. This approach can also assist in artificial evolution of enzyme catalysis from protein templates without bias for predefined catalytic sites. The prevalence of covalent intermediates in enzymatic mechanisms suggests the universal utility of the covalent complex as the basis for selection. Covalent selection by phosphonate ester exchange was applied to a phage display library of antibody variable fragments (scFv) to sample the scope and mechanism of chemical reactivity in a naive molecular library. Selected scFv segregated into structurally related covalent and noncovalent binders. Clones that reacted covalently utilized tyrosine residues exclusively as the nucleophile. Two motifs were identified by structural analysis, recruiting distinct Tyr residues of the light chain. Most clones employed Tyr32 in CDR-L1, whereas a unique clone (A.17) reacted at Tyr36 in FR-L2. Enhanced phosphonylation kinetics and modest amidase activity of A.17 suggested a primitive catalytic site. Covalent selection may thus provide access to protein molecules that approximate an early apparatus for covalent catalysis.     

Ratiometric fluorescence detection of a tag fused protein using the dual-emission artificial molecular probe

We have successfully developed a ratiometric detection system for protein of interest using the complementary recognition pair of the tetra-aspartate peptide tag and the SNARF-appended Zn(ii)-DpaTyr probe.     

Reversible covalent inhibition of a protein target

Electron-deficient Michael acceptors can be used in reversible addition/elimination reactions with thiols. These electrophiles were used to convert a known irreversible kinase inhibitor into a covalent and reversible inhibitor. Such an approach might provide high-affinity binders with increased selectivity without the toxicological risks associated with irreversible protein modifications.     

Real-time measurements of protein dynamics using fluorescence activation-coupled protein labeling method

We present a fluorescence activation-coupled protein labeling (FAPL) method, which employs small-molecular probes that exhibit almost no basal fluorescence but acquire strong fluorescence upon covalent binding to tag-proteins. This method enables real-time imaging of protein labeling without any washout process and is uniquely suitable for real-time imaging of protein dynamics on the cell surface. We applied this method to address the spatiotemporal dynamics of the EGF receptor during cell migration.     

Non-enzymatic electrochemical immunoassay using noble metal nanoparticles: a review

Electrochemical immunodetection has attracted considerable attention due to its high sensitivity, low cost and simplicity. Large efforts have recently made in order to design ultrasensitive assays. Noble metal nanoparticles (NM-NPs) offer advantages such as high conductivity and large surface-to-volume ratio. NM-NPs therefore are excellent candidates for developing electrochemical platforms for immunodetection and as signal tags. The use of biofunctionalized NM-NPs often results in amplified recognition via stronger loading of signal tags, and also in enhanced signal. This review (with 87 references) gives an overview on the current state in the use of NM-NPs in Non-enzymatic electrochemical immunosensing. We discuss the application of NM-NPs as electrode matrices and as electroactive labels (either as a carrier or as electrocatalytic labels), and compare the materials (mainly nanoparticles of gold, platinum, or of bimetallic materials) in terms of performance (for example by increasing sensitivity via label amplification or via high densities of capture molecules). A conclusion covers current challenges and gives an outlook. Rather than being exhaustive, the review focuses on representative examples that illustrate novel concepts and promising applications. NM-NPs based immunosensing opens a series of concepts for basic research and offers new tools for determination of trace amounts of protein-related analytes in environment and clinical applications.

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Absolute protein quantification by LC-ICP-MS using MeCAT peptide labeling

Nowadays, the most common strategies used in quantitative proteomics are based on isotope-coded labeling followed by specific molecule mass spectrometry. The implementation of inductively coupled plasma mass spectrometry (ICP-MS) for quantitative purposes can solve important drawbacks such as lack of sensitivity, structure-dependent responses, or difficulties in absolute quantification. Recently, lanthanide-containing labels as metal-coded affinity tag (MeCAT) reagents have been introduced, increasing the interest and scope of elemental mass spectrometry techniques for quantitative proteomics. In this work one of the first methodologies for absolute quantification of peptides and proteins using MeCAT labeling is presented. Liquid chromatography (LC) interfaced to ICP-MS has been used to separate and quantify labeled peptides while LC coupled to electrospray ionization mass spectrometry served for identification tasks. Synthetic-labeled peptides were used as standards to calibrate the response of the detector with compounds as close as possible to the target species. External calibration was employed as a quantification technique. The first step to apply this approach was MeCAT-Eu labeling and quantification by isotope dilution ICP-MS of the selected peptides. The standards were mixed in different concentrations and subjected to reverse-phase chromatography before ICP-MS detection to consider the column effect over the peptides. Thus, the prepared multi-peptide mix allowed a calibration curve to be obtained in a single chromatographic run, correcting possible non-quantitative elutions of the peptides from the column. The quantification strategy was successfully applied to other labeled peptides and to standard proteins such as digested lysozyme and bovine serum albumin.     

Non-enzymatic detection of hydrogen peroxide using a functionalized three-dimensional graphene electrode

A novel three-dimensional (3D) electrochemical sensor was developed for highly sensitive detection of hydrogen peroxide (H₂O₂). Monolithic and macroporous graphene foam grown by chemical vapor deposition (CVD) served as the electrode scaffold. Using in-situ polymerized polydopamine as the linker, the 3D electrode was functionalized with thionine molecules which can efficiently mediate the reduction of H₂O₂ at close proximity to the electrode surface. Such stable non-enzymatic sensor is able to detect H₂O₂ with a wide linear range (0.4 to 660 μM), high sensitivity (169.7 μA mM^− 1), low detection limit (80 nM), and fast response (reaching 95% of the steady current within 3 s). Furthermore, this sensor was used for real-time detection of dynamic release of H₂O₂ from live cancer cells in response to a pro-inflammatory stimulant.     

Non-enzymatic glucosylation of protein, a new method for the estimation of protein half-life in vivo

Conclusion The covalent attachment of glucose to proteins may be regarded as endogenous label of proteins. We have made use of this label to estimate the half-life of the proteins. The validity of this new approach has been demonstrated by comparing values for protein half-life found with the described method and those found in literature. Furthermore simulation of glycation of albumin in vitro yields values found in normal subjects.

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Nichtenzymatische Glucosylierung von Proteinen, eine neue Möglichkeit zur Bestimmung der Proteinhalbwertszeit

     

Highly oriented surface-growth and covalent dye labeling of mesoporous metal-organic frameworks

Mesoporous amino-functionalized metal-organic framework thin films with the UiO-68 topology were grown in a highly oriented fashion on two different self-assembled monolayers on gold. The oriented MOF films were covalently modified with the fluorescent dye Rhodamine B inside the pore system, as demonstrated with size-selective fluorescence quenching studies. Our study suggests that mesoporous metal-organic frameworks are promising hosts for the covalent attachment of numerous functional moieties in a molecularly designed crystalline environment.     

Selective labeling of polypeptides using protein farnesyltransferase via rapid oxime ligation

An aldehyde-containing alternative substrate for protein farnesyltransferase was prepared and shown to be enzymatically incorporated into a peptide and a protein. The protein was subsequently immobilized onto aminooxy-functionalized agarose beads or labeled with a fluorophore. This method for protein modification provides an alternative to the commonly employed Cu(I)-catalyzed click reaction.     

蛋白质的同时双重标记

目前,蛋白质亲和色谱在生命科学中具有很多的实际应用,但也面临着许多挑战。例如,一般化学固定方法是随机地与蛋白质各个位置的氨基或羧基反应,可能会破坏蛋白质的天然结构或其功能,并且反应的计量难以控制。因此蛋白质的定向固定化是一种较为理想的选择。但是,如何实现蛋白质稳定、持久的定向固定化?固定在非生理表面的蛋白质易于丧失其天然结构,从而失去其特有的生理功能。如何在色谱填料表面维持固定化后蛋白质的生理功能? 另外,细胞内许多蛋白质是以复合物乃至复杂的组装体形式存在,是否可以将蛋白质的复合物或组装体实现定向固定化? 对于酶的固定化也存在以上的问题。但是,在许多具体酶应用例子中,需要多种酶的参与。因此,需要发展同时固定多种酶的方法也是色谱研究的重要内容。但如何实现多种酶的同时、可控的固定化仍是一个难题。我在这里介绍的三元正交概念以及蛋白质同时双重标记方法也许会对以上问题的解决提供一种新的思路。另外,可以预期,三元正交试剂和同时双重标记在蛋白质组学的研究中也会有新的应用。     

Direct site-selective covalent protein immobilization catalyzed by a phosphopantetheinyl transferase

Immobilization of proteins onto solid supports is important in the preparation of functional protein microarrays and in the development of bead-based bioassays, biosensors, and industrial biocatalysts. In order to generate the stable, functional, and homogeneous materials required for these applications, attention has focused on methods that enable the efficient and site-specific covalent immobilization of recombinant proteins onto a wide range of platforms. To this end, the phosphopantetheinyl transferase Sfp was employed to catalyze the direct immobilization of recombinant proteins bearing the small, genetically encoded ybbR tag onto surfaces functionalized with CoA. Using mass spectrometry, it was shown that the Sfp catalyzes immobilization of a model acyl carrier protein (ACP) onto CoA-derivatized PEGA resin beads through specific covalent bond formation. Luciferase (Luc) and glutathione-S-transferase (GST) ybbR-fusion proteins were similarly immobilized onto PEGA resin retaining high levels of enzyme activity. This strategy was also successfully applied for the immobilization of the ACP, as well as ybbR-Luc, -GST, and -thioredoxin fusion proteins, on hydrogel microarray slides. Overall, the Sfp-catalyzed surface ligation is mild, quantitative, and rapid, occurring in a single step without prior chemical modification of the target protein. Immobilization of the target proteins directly from a cell lysate mixture was also demonstrated.