A simple method for electrophilic functionalization of DNA

[reaction: see text] An extremely simple and versatile method for placing an electrophilic functional group (iodide) at the 5' end of oligodeoxyribonucleotides is described. The reaction is carried out while the protected oligodeoxyribonucleotide remains on a solid support and utilizes inexpensive iodination chemistry. We demonstrate that this reaction can be automated on a DNA synthesizer as the last step of DNA synthesis.     

A simple MALDI target plate with channel design to improve detection sensitivity and reproducibility for quantitative analysis of biomolecules

Overcoming the detrimental effects of sweet spots during crystallization is an important step to improve the quantitative abilities of matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry. In this study, we introduce MALDI targets, which exhibit a channel design to reduce sweet spot phenomena and improve reproducibility. The size of the channels was 3.0 mm in length, 0.35 mm in depth, and 0.40 mm in width, adjusted to the width of the implemented laser beam. For sample deposition, the matrix/sample mixture was homogenously deposited into the channels using capillary action. To demonstrate the proof‐of‐principle, the novel plates were used for the quantification of acetyl‐L‐carnitine in human blood plasma using a combined standard addition and isotope dilution method. The results showed that the reproducibility of acetyl‐L‐carnitine detection was highly improved over a conventional MALDI‐MS assay, with RSD values of less than 5.9% in comparison with 15.6% using the regular MALDI method. The limits of quantification using the new plates were lowered approximately two‐fold in comparison with a standard rastering approach on a smooth stainless‐steel plate. Matrix effects were also assessed and shown to be negligible. The new assay was subsequently applied to the quantification of acetyl‐L‐carnitine in human plasma samples.     

A simple method for functionalization of cellulose membrane for covalent immobilization of biomolecules

Activated cellulose membrane was prepared by a simple photochemical reaction at 365 nm in 12 min using a photolinker, 1-fluoro-2-nitro-4-azidobenzene. XPS analysis of the activated cellulose membrane confirmed the presence of nitrogen and fluorine in the ratio of 2:1. Immobilization of a protein molecule onto the activated membrane occurred in 2 h at 37 °C. In contrast, no appreciable immobilization occurred onto the untreated surface. Disappearance of the fluorine peak in the XPS spectra of membrane having immobilized HRP confirmed covalent binding of the protein onto the activated membrane. Invertase was also immobilised onto the activated membrane and used in a flow through reactor system for conversion of sucrose to glucose and fructose. Immobilized invertase was found to be stable for at least 72 h of continuous run. The kinetic parameters of the enzyme reaction, Michaelis constant (K_m) and V_max value of immobilized invertase was studied. The activated membrane when used in an ELISA procedure to detect immunoglobulins in human sera, showed around 2.6-fold higher sensitivity than the untreated membrane. The activated cellulose membrane has the potential for versatile applications such as in diagnostics, in flow reactor system for an enzyme-catalysed reaction and in membrane based affinity chromatography.     

A simple method for the preparation and selective functionalization of 4,5-diaminopyrazoles

A simple procedure for the synthesis and further functionalization of 4,5-diaminopyrazoles using mild conditions is reported herein. The desired products were obtained in good yield, and the structures have been confirmed by X-ray crystallography.     

A novel,simple and sensitive ligand affinity capture method for detecting molecular interactions by MALDI mass spectrometry

A simple and sensitive ligand affinity capture method (LAC) was developed to detect biotinylated biomolecules bound to a biotin–avidin base by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI ToF MS). Glass slides covered with a metal film for MALDI MS applications were treated with amino‐silane and derivatized with biotin followed by binding of avidin. Washing buffers with high ionic strength increased the specificity of the subsequent binding of biotinylated biomolecules to the avidin layer. A combined thin layer‐dried droplet method using α‐cyano‐4‐hydroxycinnamic acid (CHCA) in acetone or ethyl acetate resulted in the most intense ions of biotinylated polymyxin B, whereas the matrix conditions did not influence the detection of angiotensin II. Addition of biotinylated biomolecules in the low femtomole to low picomole range resulted in sufficient ion intensity for detection by the LAC method. The LAC concept was extended by binding of biotinylated lipopolysaccharide to the biotin–avidin base followed by preferential capture and specific detection of the binding antagonist polymyxin B. Copyright © 2008 John Wiley & Sons, Ltd.     

A novel multi-enzymatic high throughput assay for transaminase activity

Aminotransferases (ATs) are crucial enzymes for prokaryote and eukaryote metabolism, and have attracted industrial attention for large scale synthesis of amino acids and chiral amines.A high throughput screening procedure for branched chain aminotransferases (BCATs) was developed to identify optimal expression on a large number of expression clones. Escherichia coli BCAT, encoded by the ilvE gene, was expressed in E. coli and Pichia pastoris. This simple colorimetric assay procedure allowed the identification of optimal clones for ilvE expression and enabled the testing of its activity in cell lysates or on whole cell catalysis.     

A simple approach to covalent functionalization of boron nitride nanotubes

A novel and simple method for the preparation of chemically functionalized boron nitride nanotubes (BNNTs) is presented. Thanks to a strong oxidation followed by the silanization of the surface through 3-aminopropyl-triethoxysilane (APTES), BNNTs exposing amino groups on their surface were successfully obtained. The efficacy of the procedure was assessed with EDS and XPS analyses, which demonstrated a successful functionalization of ~15% boron sites. This approach opens interesting perspectives for further modification of BNNTs with several kinds of molecules. Since, in particular, biomedical applications are envisaged, we also demonstrated in vitro biocompatibility and cellular up-take of the functionalized BNNTs.     

A simple tagging system for protein encapsulation

Molecular containers that encapsulate specific cargo can be useful for many natural and non-natural processes. We report a simple system, based on charge complementarity, for the encapsulation of appropriately tagged proteins within an engineered, proteinaceous capsid. Four negative charges per monomer were added to the lumazine synthase from Aquifex aeolicus (AaLS). The capsids formed by the engineered AaLS associate with green fluorescent protein bearing a positively charged deca-arginine tag upon coproduction in Escherichia coli. Analytical ultracentrifugation and scanning force microscopy studies indicated that the engineered AaLS retains the ability to form capsids, but that their average size was substantially increased. The success of this strategy demonstrates that both the container and guest components of protein-based encapsulation systems can be convergently designed in a straightforward manner, which may help to extend their versatility.     

A new, simple approach to confer permanent antimicrobial properties to hydroxylated surfaces by surface functionalization

A new, simple method to obtain ultrathin polycationic monolayers on hydroxylated surfaces is described which uses a bifunctional copolymer comprising a reactive part (trimethoxysilane) and positive charges (quaternary ammonium salts) to confer antimicrobial properties.     

Organotin and organogermanium linkers for simple, direct functionalization of polyoxotungstates

Fifteen Keggin-anion-derived polytungstates [TW11O39[MCH2CH2X]]n- (T = Si, Ge, Ga; M = Sn, Ge; X = COOH, COOCH3, CONH2, CN; n = 5, 6) were prepared in aqueous or aqueous-organic solution from the corresponding lacunary polytungstates and trichlorotin and -germanium precursors, and were isolated as caesium salts. The derivatized polytungstates were characterized by elemental analysis, multinuclear NMR spectroscopy, and cyclic voltammetry; they are stable in aqueous solution to pH 6-7. NMR spectroscopy revealed the presence of a second (beta1 or beta3) isomer in the tungstogallate derivatives. Acid hydrolysis of the ester and nitrile derivatives could be achieved without decomposition of the polytungstate moieties, and esterification and amidation of the carboxylate functions was straightforward using standard coupling techniques, e.g. the formation, isolation and characterization of [SiW11O39[Ge(CH2)2CONHCH2COOCH3]]5- from glycine methyl ester. Since the Cl3MCH2CH2X precursors are readily accessible by hydrostannation/germanation reactions with the corresponding alkenes, novel coupled polytungstates, such as [(SiW11O39GeCH2CH2COOCH2)4C]20- from pentaerythritol tetraacrylate, can also be prepared.     

Sequence dependent fragmentation of peptides generated by MALDI quadrupole time-of-flight (MALDI Q-TOF) mass spectrometry and its implications for protein identification

A study has been undertaken to evaluate the usefulness of MALDI Q-TOF data for protein identification. The comparison of MS data of protein digests obtained on a conventional MALDI TOF instrument to the MS data from the MALDI Q-TOF reveal peptide patterns with similar intensity ratios. However, comparison of MS/MS Q-TOF data produced by nanoelectrospray versus MALDI reveals striking differences. Peptide fragment ions obtained from doubly charged precursors produced by nanoelectrospray are mainly y-type ions with some b-ions in the lower mass range. In contrast, peptide fragment ions produced from the singly charged ions originating from the MALDI source are a mixture of y-, b- and a-ions accompanied by ions resulting from neutral loss of ammonia or water. The ratio and intensity of these fragment ions is found to be strongly sequence dependent for MALDI generated ions. The singly charged peptides generated by MALDI show a preferential cleavage of the C-terminal bond of acidic residues aspartic and glutamic acid and the N-terminal bond of proline. This preferential cleavage can be explained by the mobile proton model and is present in peptides that contain both arginine and an acidic amino acid. The MALDI Q-TOF MS/MS data of 24 out of 26 proteolytic peptides produced by trypsin or Asp-N digestions were successfully used for protein identification via database searching, thus indicating the general usefulness of the data for protein identification. De novo sequencing using a mixture of 160/18O water during digestion has been explored and de novo sequences for a number of peptides have been obtained.     

单克隆抗体1F2的制备、纯化及质谱测定

用小分子meso -四 (α,α ,α ,α -O -苯乙酰苯 )卟啉免疫Balb/c小鼠 ,用单克隆抗体技术得到细胞株 1F2和单抗 1F2 ,这是对传统免疫理论的一个突破 .利用高效液相色谱和基质辅助激光解吸质谱 (matrixassistedlaserdesorptionionizationtime_of_flightmassspectography ;缩写为MALDI/TOFMS)证明了纯化得到单抗体 1F2的纯度很好 ,同时得到的单抗 1F2的相对分子量为 15 6 6 78.8Da .MALDI/TOFMS提供了一种测定蛋白质相对分子量和纯度快速准确的方法     

A simple method for aligning many protein sequences

A simple extension of the Needleman and Wunsch algorithm for aligning pairs of protein sequences allows it to be used for the efficient generation of very large multiple-sequence alignments whose members are similar. This technique could have applications in a broad range of high-volume genomics projects.     

A simple synthesis for 2-acetonylcyclododecanone

A simple method is proposed for the alkylation of cyclododecanone by propargyl halides under phase transfer catalysis conditions with the formation of 2-propargylcyclododecanone. The hydration of 2-propargylcyclododecanone upon catalysis by mercury compounds leads to either 14-methyl-13-oxabicyclo[10.3.0]-pentadeca-1(12),14-diene or 2-acetonylcyclododecanone depending on the reaction conditions. Both these compounds are also readily obtained from 2-(2-chloropropen-2-yl)cyclododecanone which readily forms upon the alkylation of cyclododecanone by 1,2-dichloropropene under phase transfer catalysis conditions.Translated from Izvestiya Akademil Nauk SSSR, Seriya Khimicheskaya, No. 4, pp. 963–965, April, 1991.     

A furoindoline synthesis by remote radical functionalization

The preliminary results of an investigation toward a synthesis of furoindolines from 3-(2-hydroxyethyl)indolines by remote radical functionalization are described. Using an oxidative radical cyclization, it was discovered that the intramolecular hydrogen abstraction was only successful when the resulting radical (and hence carbocation) was resonance stabilized by adjacent tertiary amine and phenyl groups. The successful cyclization affords diastereomeric furoindolines, one of which contains a highly strained trans-fused 5,5-ring system. This furoindoline synthesis contains a rare example of an alkoxy radical promoted hydrogen atom transfer of a proton attached to a nitrogen-substituted carbon.     

A simple protocol for protein extraction of recalcitrant fruit tissues suitable for 2-DE and MS analysis

Fruit tissues are considered recalcitrant plant tissue for proteomic analysis. Three phenol-free protein extraction procedures for 2-DE were compared and evaluated on apple fruit proteins. Incorporation of hot SDS buffer, extraction with TCA/acetone precipitation was found to be the most effective protocol. The results from SDS-PAGE and 2-DE analysis showed high quality proteins. More than 500 apple polypeptides were separated on a small scale 2-DE gel. The successful protocol was further tested on banana fruit, in which 504 and 386 proteins were detected in peel and flesh tissues, respectively. To demonstrate the quality of the extracted proteins, several protein spots from apple and banana peels were cut from 2-DE gels, analyzed by MS and have been tentatively identified. The protocol described in this study is a simple procedure which could be routinely used in proteomic studies of many types of recalcitrant fruit tissues.     

Impact of un-polymerized acrylamide monomer residues onto protein identification by MALDI TOF MS

Polyacrylamide gel electrophoresis is a powerful tool for protein mixture separation frequently used in proteomic studies. This article describes the problems which can arise during the identification process of separated proteins by matrix assisted laser desorption/ionization mass spectrometry. Presence of residual acrylamide monomers in the peptide samples leads to a significant peptide signal suppression. In the case of protein prohibitin, isolated from the MCF 7 breast cancer cell line, the peptide signal suppression was observed in 50% of all detected peptides.      

A simple method for improving protein solubility and long-term stability

Increasing a protein concentration in solution to the required level, without causing aggregation and precipitation is often a challenging but important task, especially in the field of structural biology; as little as 20% of nonmembrane proteins have been found to be suitable candidates for structural studies predominantly due to poor protein solubility. We demonstrate here that simultaneous addition of charged amino acids L-Arg and L-Glu at 50 mM to the buffer can dramatically increase the maximum achievable concentration of soluble protein (up to 8.7 times). These amino acids are effective in preventing protein aggregation and precipitation, and they dramatically increase the long-term stability of the sample; additionally, they protect protein samples from proteolytic degradation. Specific protein-protein and protein-RNA interactions are not adversely affected by the presence of these amino acids. These additives are particularly suitable for situations where high protein concentration and long-term stability are required, including solution-state studies of isotopically labeled proteins by NMR.     

A novel route to substituted poly(vinyl pyrrolidone)s via simple functionalization of 1-vinyl-2-pyrrolidone in the 3-position by ring-opening reactions

A general synthetic approach is described that allows to obtain novel 1-vinyl-2-pyrrolidone (VP) derivatives with different kinds of functional groups in its 3-position in a one-pot reaction. The strategy used to achieve this goal is the reaction of the carboxamide anion of 1-vinyl-2-pyrrolidone with cyclic precursors of these functionalities. While the driving force for the reactions of three-membered rings is their high annular tension, it is shown here that larger heterocycles can be opened with good yield when additional electron-withdrawing groups are present in the ring. This leads to VP with longer spacer groups between the functionality and the future polymeric main chain. Furthermore, the high versatility of this procedure is demonstrated by the preparation, for the first time, of VP modified with amine or sulfonic functionalities that allow to prepare the corresponding aminated and sulfonated PVPs.