2DE‐based approach for estimation of number of protein species in a cell

Insufficient sensitivity of methods for detection of proteins at a single molecule level does not yet allow obtaining the whole image of human proteome. But to go further, we need at least to know the proteome size, or how many different protein species compose this proteome. This is the task that could be at least partially realized by the method described in this article. The approach used in our study is based on detection of protein spots in 2DE after staining by protein dyes with various sensitivities. As the different protein spots contain different protein species, counting the spots opens a way for estimation of number of protein species. The function representing the dependence of the number of protein spots on sensitivity or LOD of protein dyes was generated. And extrapolation of this function curve to theoretical point of the maximum sensitivity (detection of a single smallest polypeptide) allowed to counting the number of different molecules (polypeptide species) at the concentration level of a single polypeptide per proteome. Using this approach, it was estimated that the minimal numbers of protein species for model objects, Escherichia coli and Pirococcus furiosus, are 6200 and 3400, respectively. We expect a single human cell (HepG2) to contain minimum 70 000 protein species.     

Pairwise decomposition of an MMGBSA energy function for computational protein design

Computational protein design (CPD) aims at predicting new proteins or modifying existing ones. The computational challenge is huge as it requires exploring an enormous sequence and conformation space. The difficulty can be reduced by considering a fixed backbone and a discrete set of sidechain conformations. Another common strategy consists in precalculating a pairwise energy matrix, from which the energy of any sequence/conformation can be quickly obtained. In this work, we examine the pairwise decomposition of protein MMGBSA energy functions from a general theoretical perspective, and an implementation proposed earlier for CPD. It includes a Generalized Born term, whose many‐body character is overcome using an effective dielectric environment, and a Surface Area term, for which we present an improved pairwise decomposition. A detailed evaluation of the error introduced by the decomposition on the different energy components is performed. We show that the error remains reasonable, compared to other uncertainties. © 2014 Wiley Periodicals, Inc.     

基于微流控技术的蛋白质结晶及其筛选方法的研究进展简

微流控技术以其高通量、低消耗和集成化等优点成为蛋白质结晶微型化研究的重要手段. 本文综述了基于微流控技术的蛋白质结晶技术和方法,主要包括微泵微阀、液滴(Droplet)、滑动芯片(SlipChip)以及液滴实验室(DropLab)等技术. 此外,还针对当前膜蛋白在结构生物学研究中的重要地位,综述了应用于膜蛋白结晶的微流控技术的研究进展.     

基于多肽的电化学传感器在蛋白质检测中应用的研究进展

生物体内的细胞通常会分泌各种各样的蛋白质,这些蛋白质在生物体中发挥着重要作用,尤其是可被用于诊断各种疾病的发生和发展。多肽具有良好选择性、空间适应能力和识别灵活的特点,可与不同类型的蛋白分子形成非共价键,用于蛋白质的生物检测。将多肽与电化学生物传感器结合用于蛋白质的广谱检测具有良好的发展前景。本文介绍了多肽修饰的电化学传感器在不同蛋白质检测方面的研究进展,分析了待测蛋白质的不同对多肽修饰的电化学传感器分类的影响及其优缺点,提出了基于多肽的电化学传感器在不同蛋白质检测中存在的问题,并展望了其未来发展。     

Improved Stabilities of Labeling Probes for the Selective Modification of Endogenous Proteins in Living Cells and In Vivo

To date, various affinity-based protein labeling probes have been developed and applied in biological research to modify endogenous proteins in cell lysates and on the cell surface. However, the reactive groups on the labeling probes are also the cause of probe instability and nonselective labeling in a more complex environment, e. g., intracellular and in vivo. Here, we show that labeling probes composed of a sterically stabilized difluorophenyl pivalate can achieve efficient and selective labeling of endogenous proteins on the cell surface, inside living cells and in vivo. As compared with the existing protein labeling probes, probes with the difluorophenyl pivalate exhibit several advantages, including long-term stability in stock solutions, resistance to enzymatic hydrolysis and can be customized easily with diverse fluorophores and protein ligands. With this probe design, endogenous hypoxia biomarker in living cells and nude mice were successfully labeled and validated by in vivo, ex vivo, and immunohistochemistry imaging.     

One-Pot and Two-Chamber Methodologies for Using Acetylene Surrogates in the Synthesis of Pyridazines and Their D-Labeled Derivatives

Acetylene surrogates are efficient tools in modern organic chemistry with largely unexplored potential in the construction of heterocyclic cores. Two novel synthetic paths to 3,6-disubstituted pyridazines were proposed using readily available acetylene surrogates through flexible C₂ unit installation procedures in a common reaction space mode (one-pot) and distributed reaction space mode (two-chamber): (1) an interaction of 1,2,4,5-tetrazine and its acceptor-functionalized derivatives with a CaC₂−H₂O mixture performed in a two-chamber reactor led to the corresponding pyridazines in quantitative yields; (2) [4+2] cycloaddition of 1,2,4,5-tetrazines to benzyl vinyl ether can be considered a universal synthetic path to a wide range of pyridazines. Replacing water with D₂O and vinyl ether with its trideuterated analog in the developed procedures, a range of 4,5-dideuteropyridazines of 95–99% deuteration degree was synthesized for the first time. Quantum chemical modeling allowed to quantify the substituent effect in both synthetic pathways.     

18O/16O-Encoding Strategy for Microscale Stereochemical Determination of Peptidic Natural Products

The demand for more efficient methods of establishing the undetermined stereochemistries of peptidic natural products continues unabated. A new method for microscale stereochemical determination was devised by integrating solid-phase synthesis, split-and-mix randomization, ¹⁸O/¹⁶O-encoding of d /l -configurations, tandem mass spectrometry, and biological evaluation. Here we applied gramicidin A as the molecule for a blind test. Gramicidin A and its 31 diastereomers were randomly prepared in microgram scale with ¹⁸O/¹⁶O-stereochemical encoding and subjected to MS/MS-structural determination and cytotoxicity assay. Only the parent gramicidin A was selected from among the 32 stereoisomers, validating the high reliability of the present strategy.     

Exploiting Protein N-Terminus for Site-Specific Bioconjugation

Although a plethora of chemistries have been developed to selectively decorate protein molecules, novel strategies continue to be reported with the final aim of improving selectivity and mildness of the reaction conditions, preserve protein integrity, and fulfill all the increasing requirements of the modern applications of protein conjugates. The targeting of the protein N-terminal alpha-amine group appears a convenient solution to the issue, emerging as a useful and unique reactive site universally present in each protein molecule. Herein, we provide an updated overview of the methodologies developed until today to afford the selective modification of proteins through the targeting of the N-terminal alpha-amine. Chemical and enzymatic strategies enabling the selective labeling of the protein N-terminal alpha-amine group are described.