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.     

Convenient one-pot procedure for synthesizing 4,4′-dimethoxy-boradiaza-s-indacene dyes and their application to cell labeling

We succeeded in developing a convenient one-pot pathway for synthesizing 4,4′-dimethoxy-boradiaza-s-indacene dyes 4a-d. The structures feature two methoxy groups in place of the fluorine atoms in 4,4′-difluoro-4-boradiaza-s-indacene. These novel dyes emitted green fluorescence and possessed moderate to high fluorescence quantum yields (Φ=0.32-0.93). We demonstrated that these dyes have applicability to cell labeling.     

Water-soluble NIR fluorescent probes based on squaraine and their application for protein labeling.

Water-soluble near-infrared (NIR) fluorescent labeling probes, named KSQ-3 and -4, which are based on a squaraine backbone, were synthesized and applied to biological labeling. The presented results demonstrate that the large, planar and hydrophobic squaraine dye becomes fully soluble in aqueous solution by the introduction of several sulfo group terminated alkyl substituents. Especially KSQ-4, which is substituted with four sulfo groups, exhibited perfect water solubility and significant fluorescence emission at the NIR region (817 nm) in the presence of bovine serum albumin (BSA). BSA was covalently labeled with KSQ-4, and the conjugate showed a strong absorption peak at 787 nm, which indicates compatibility with commercially available NIR laser diodes used for exciting the fluorophore. Furthermore, strong fluorescence emission was observed at 812 nm (phi = 0.08).     

Advances in polymer and polymeric nanostructures for protein conjugation

Linear polymers have been considered the best molecular structures for the formation of efficient protein conjugates due to their biological advantages, synthetic convenience and ease of functionalization. In recent years, much attention has been dedicated to develop synthetic strategies that produce the most control over protein conjugation utilizing linear polymers as scaffolds. As a result, different conjugate models, such as semitelechelic, homotelechelic, heterotelechelic and branched or star polymer conjugates, have been obtained that take advantage of these well-controlled synthetic strategies. Development of protein conjugates using nanostructures and the formation of said nanostructures from protein–polymer bioconjugates are other areas in the protein bioconjugation field. Although several polymer–protein technologies have been developed from these discoveries, few review articles have focused on the design and function of these polymers and nanostructures. This review will highlight some recent advances in protein-linear polymer technologies that employ protein covalent conjugation and successful protein-nanostructure bioconjugates (covalent conjugation as well) that have shown great potential for biological applications.     

Ionic liquid/mesoporous carbon/protein composite microelectrode and its biosensing application

A functional composite was prepared by mixing mesoporous carbon, glucose oxidase (GOD) and 1-butyl-3-methylimidazolium hexafluorophosphate, an ionic liquid, and characterized by SEM and RA-IR. The composite was filled in a microcavity to fabricate a paste microelectrode, demonstrating direct electrochemistry of GOD with a pair of well-defined redox peaks. The composite microelectrode was used as a glucose microsensor, showing good sensitivity over a concentration range from 10.0 to 80.0 μmol/L and a Michaelis–Menten constant of 2.42 μmol/L. This work demonstrates an efficient and accurate approach to study direct electrochemistry with potential applications in various enzymatic biosensors.     

A proximity labeling method for protein–protein interactions on cell membrane

Antibodies targeting specific antigens are widely utilized in biological research to investigate protein interactions or to quantify target antigens. Here, we introduce antigen–antibody proximity labeling (AAPL), a novel method to map the antigen interaction sites as well as interactors of antibody-targeted proteins. As a proof of concept, AAPL was demonstrated using sodium/potassium transporting ATPase (ATP1A1) and epidermal growth factor receptor 2 (ERBB2)-specific antibodies that were modified with an Fe(iii) catalytic probe. Once bound to their target proteins, Fe(iii)-induced catalytic oxidation occurred in proximity of the antigen''s epitope. Oxidative proteomic analysis was then used to determine the degree of oxidation, the site of oxidation within the targeted antigen, and the interacting proteins that were in close proximity to the targeted antigen. An AAPL score was generated for each protein yielding the specificity of the oxidation and proximity of the interacting protein to the target antigen. As a final demonstration of its utility, the AAPL approach was applied to map the interactors of liver–intestine-cadherin (CDH17) in colon cancer cells.

Modified catalytic antibodies targeting specific antigens are employed to investigate protein interactions and antigen interaction sites.     

In-gel protein N- and C-termini identification and its application for transgenic protein characterization

A new method for the determination of N- and C-termini of a protein isolated in a polyacrylamide gel is introduced. In-gel partial protein hydrolysis by hydrochloric acid is used to generate N- and C-terminal peptides for identification. This new method is complementary to existing techniques. The application of the in-gel protein termini identification technique to the characterization of the transgenic protein diacylglycerol acyltransferase (UrDGAT2A) purified from soybean seeds is also reported here. Both N- and C-termini of UrDGAT2A were successfully identified and the N-terminus was found to be blocked by acetylation. The analysis results of UrDGAT2A and two commercial proteins (bovine serum albumin (BSA) and alcohol dehydrogenase) are used to demonstrate the effectiveness of the method in identifying actual N- and C-termini, terminal truncation and blocking.     

No-wash protein labeling with designed fluorogenic probes and application to real-time pulse-chase analysis

Small molecule labeling techniques for cellular proteins under physiological conditions are very promising for revealing new biological functions. We developed a no-wash fluorogenic labeling system by exploiting fluorescence resonance energy transfer (FRET)-based fluorescein-cephalosporin-azopyridinium probes and a mutant β-lactamase tag. Fast quencher elimination, hydrophilicity, and high resistance against autodegradation were achieved by rational refinement of the structure. By applying the probe to real-time pulse-chase analysis, the trafficking of epidermal growth factor receptors between cell surface and intracellular region was imaged. In addition, membrane-permeable derivatization of the probe enabled no-wash fluorogenic labeling of intracellular proteins.     

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。     

Inverse 15N-metabolic labeling/mass spectrometry for comparative proteomics and rapid identification of protein markers/targets

The inverse labeling/mass spectrometry strategy has been applied to protein metabolic (15)N labeling for gel-free proteomics to achieve the rapid identification of protein markers/targets. Inverse labeling involves culturing both the perturbed (by disease or by a drug treatment) and control samples each in two separate pools of normal and (15)N-enriched culture media such that four pools are produced as opposed to two in a conventional labeling approach. The inverse labeling is then achieved by combining the normal (14)N-control with the (15)N-perturbed sample, and the (15)N-control with the (14)N-perturbed sample. Both mixtures are then proteolyzed and analyzed by mass spectrometry (coupled with on-line or off-line separation). Inverse labeling overcomes difficulties associated with protein metabolic labeling with regard to isotopic peak correlation and data interpretation in the single-experiment approach (due to the non-predictable/variable mass difference). When two data sets from inverse labeling are compared, proteins of differential expression are readily recognized by a characteristic inverse labeling pattern or apparent qualitative mass shifts between the two inverse labeling analyses. MS/MS fragmentation data provide further confirmation and are subsequently used to search protein databases for protein identification. The methodology has been applied successfully to two model systems in this study. Utilizing the inverse labeling strategy, one can use any mass spectrometer of standard unit resolution, and acquire only the minimum, essential data to achieve the rapid and unambiguous identification of differentially expressed protein markers/targets. The strategy permits quick focus on the signals of differentially expressed proteins. It eliminates the detection ambiguities caused by the dynamic range of detection. Finally, inverse labeling enables the detection of covalent changes of proteins responding to a perturbation that one might fail to distinguish with a conventional labeling experiment.     

SNAP-tag fluorogenic probes for wash free protein labeling

In this review, we described the design strategies of SNAP-tag fl uorogenic probes with turn-on fl uorescence responses, which minimized the fl uorescence background and allowed for direct imaging in living cells without wash-out steps. These probes can apply in real-time analysis of protein localization, dynamics, and protein– protein interactions in living cells. Furthermore, the excellent fl uorescent properties made it possible to apply some of the probes in super-resolution fl uorescence imaging.     

A novel pyrimidine-based stable-isotope labeling reagent and its application to quantitative analysis using matrix-assisted laser desorption/ionization mass spectrometry

As an extension of our previous work, a novel pyrimidine-based stable-isotope labeling reagent, [d(0)]-/[d(6)]-4,6-dimethoxy-2-(methylsulfonyl)pyrimidine (DMMSP), was developed for comparative quantification of proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Our one-step labeling strategy combines several desirable properties such as cysteine-specific labeling, signal amplification and direct analysis with minimum sample handling. All these features not only allow easy interpretation for protein identification and quantification but also ensure rapid and sensitive progression to MS analysis. Using cysteine, Cys-containing peptide, and lysozyme digest as model samples, the labeling methodology was established and the following pilot application for quantitative analysis was accomplished with high confidence, accuracy, efficiency, and reproducibility. The application of DMMSP-labeling strategy is expected to provide a powerful new tool for comparative proteome research, especially for the analysis of low-abundance proteins.     

Analytical performance of reciprocal isotope labeling of proteome digests for quantitative proteomics and its application for comparative studies of aerobic and anaerobic Escherichia coli proteomes

Due to limited sample amounts, instrument time considerations, and reagent costs, only a small number of replicate experiments are typically performed for quantitative proteome analyses. Generation of reproducible data that can be readily assessed for consistency within a small number of datasets is critical for accurate quantification. We report our investigation of a strategy using reciprocal isotope labeling of two comparative samples as a tool for determining proteome changes. Reciprocal labeling was evaluated to determine the internal consistency of quantified proteome changes from Escherichia coli grown under aerobic and anaerobic conditions. Qualitatively, the peptide overlap between replicate analyses of the same sample and reverse labeled samples were found to be within 8%. Quantitatively, reciprocal analyses showed only a slight increase in average overall inconsistency when compared with replicate analyses (1.29 vs. 1.24-fold difference). Most importantly, reverse labeling was successfully used to identify spurious values resulting from incorrect peptide identifications and poor peak fitting. After removal of 5% of the peptide data with low reproducibility, a total of 275 differentially expressed proteins (>1.50-fold difference) were consistently identified and were then subjected to bioinformatics analysis. General considerations and guidelines for reciprocal labeling experimental design and biological significance of obtained results are discussed.     

化学标记与蛋白质/多肽的识别检测

化学标记技术可以实现选择性地标记蛋白质/多肽分子,从而极大地提高了对蛋白质/多肽的识别效率和检测灵敏度,是突破蛋白质/多肽化学组成局限和仪器分析检测能力瓶颈的有效途径.本文对目前这一领域的研究现状扼要地进行了综述,主要包括针对蛋白质/多肽分子中内源氨基酸残基的标记策略、蛋白质/多肽分子中翻译后修饰基团的标记策略、基因编码表达肽段的标记策略以及配体/抗体亲和标记策略.透过这些研究所取得的成果,可以断定化学标记技术将会不断发展并将在蛋白质及蛋白质组学研究中发挥重要作用.     

Comparison of continuous and pulsed labeling amide hydrogen exchange/mass spectrometry for studies of protein dynamics

In contrast to the rigid structures portrayed by X-ray diffraction, proteins in solution display constant motion which leads to populations that are momentarily unfolded. To begin to understand protein dynamics, we must have experimental methods for determining rates of folding and unfolding, as well as for identifying structures of folding and unfolding intermediates. Amide hydrogen exchange has become an important tool for such measurements. When urea is used to stabilize unfolded forms of proteins, the refolding rates may become slower than the rates of isotope exchange. In such cases, the intermolecular distribution of deuterium among the entire population of molecules may become bimodal, giving rise to a bimodal distribution of isotope peaks in mass spectra of the protein or its peptic fragments. When the protein is exposed continuously to D2O, the relative intensities of the two envelopes of isotope peaks give an integrated account of populations participating in the folding/unfolding process. However, when the protein is exposed only briefly to D2O, the relative intensities of the two envelopes of isotope peaks give an instantaneous measure of the folded/unfolded populations. Application of these two labeling methods to a large protein, aldolase, is described along with a discussion of specific parameters required to optimize these experiments.     

一种检测CO32-比率荧光探针的合成及其在细胞成像中的应用

本文设计开发了一种以2,6-二甲酰基对甲苯酚为母体的新型荧光探针HMI,可用于高效识别EtOH-H₂O (8/2, v/v, HEPES 10 mM, pH =7.4)体系中的CO_3^2-。HMI在660 nm处显示发射带,加入CO_3^2-后,在600 nm的等吸收点激发时,原来在660 nm处的荧光淬灭,而以540 nm为中心的新发射带荧光显着增加,为比率型荧光探针。HMI对CO_3^2-表现出高选择性且具有较强的抗干扰能力。此外,荧光探针HMI对CO_3^2-荧光响应的检测限较低,可达到3.938×10^_-6 M。更具有意义的是,HMI探针对CO_3^2-的检测能够在实际水样中起到很好的应用,而且细胞成像研究表明,HMI可用于活体MCF-7细胞中CO_3^2-的成像。