Sensitive targeted multiple protein quantification based on elemental detection of Quantum Dots

A generic strategy based on the use of CdSe/ZnS Quantum Dots (QDs) as elemental labels for protein quantification, using immunoassays with elemental mass spectrometry (ICP-MS), detection is presented. In this strategy, streptavidin modified QDs (QDs-SA) are bioconjugated to a biotinylated secondary antibody (b-Ab₂). After a multi-technique characterization of the synthesized generic platform (QDs-SA-b-Ab₂) it was applied to the sequential quantification of five proteins (transferrin, complement C3, apolipoprotein A1, transthyretin and apolipoprotein A4) at different concentration levels in human serum samples. It is shown how this generic strategy does only require the appropriate unlabeled primary antibody for each protein to be detected. Therefore, it introduces a way out to the need for the cumbersome and specific bioconjugation of the QDs to the corresponding specific recognition antibody for every target analyte (protein). Results obtained were validated with those obtained using UV–vis spectrophotometry and commercial ELISA Kits.     

A volatolomic approach using cerumen as biofluid to diagnose bovine intoxication by Stryphnodendron rotundifolium

An innovative volatolomic approach employs the detection of biomarkers present in cerumen (earwax) to identify cattle intoxication by Stryphnodendron rotundifolium Mart., Fabaceae (popularly known as barbatimão). S. rotundifolium is a poisonous plant with the toxic compound undefined and widely distributed throughout the Brazilian territory. Cerumen samples from cattle of two local Brazilian breeds (‘Curraleiro Pé-Duro’ and ‘Pantaneiro’) were collected during an experimental intoxication protocol and analyzed using headspace (HS)/GC–MS followed by multivariate analysis (genetic algorithm for a partial least squares, cluster analysis, and classification and regression trees). A total of 106 volatile organic metabolites were identified in the cerumen samples of bovines. The intoxication by S. rotundifolium influenced the cerumen volatolomic profile of the bovines throughout the intoxication protocol. In this way, it was possible to detect biomarkers for cattle intoxication. Among the biomarkers, 2-octyldecanol and 9-tetradecen-1-ol were able to discriminate all samples between intoxicated and nonintoxicated bovines. The cattle intoxication diagnosis by S. rotundifolium was accomplished by applying the cerumen analysis using HS/GC–MS, in an easy, accurate, and noninvasive way. Thus, the proposed bioanalytical chromatography protocol is a useful tool in veterinary applications to determine this kind of intoxication.     

Enlargement of the Wiener filtration by an absolutely continuous random variable via Malliavin’s calculus

Summary. The analytic treatment of problems related to the asymptotic behaviour of random dynamical systems generated by stochastic differential equations suffers from the presence of non-adapted random invariant measures. Semimartingale theory becomes accessible if the underlying Wiener filtration is enlarged by the information carried by the orthogonal projectors on the Oseledets spaces of the (linearized) system. We study the corresponding problem of preservation of the semimartingale property and the validity of a priori inequalities between the norms of stochastic integrals in the enlarged filtration and norms of their quadratic variations in case the random element F enlarging the filtration is real valued and possesses an absolutely continuous law. Applying the tools of Malliavin’s calculus, we give smoothness conditions on F under which the semimartingale property is preserved and a priori martingale inequalities are valid. Received: 12 April 1995 / In revised form: 7 March 1996     

A new family of proximity graphs: Class cover catch digraphs

Motivated by issues in machine learning and statistical pattern classification, we investigate a class cover problem (CCP) with an associated family of directed graphs—class cover catch digraphs (CCCDs). CCCDs are a special case of catch digraphs. Solving the underlying CCP is equivalent to finding a smallest cardinality dominating set for the associated CCCD, which in turn provides regularization for statistical pattern classification. Some relevant properties of CCCDs are studied and a characterization of a family of CCCDs is given.     

2,7-二氯荧光素与蛋白质相互作用的分光光度法研究

首次采用分光光度法研究了模拟生理条件(pH 7.4 Tris-HCl缓冲溶液,离子强度I=0.1)下2,7-二氯荧光素与牛血清白蛋白(BSA)相互作用的情况.研究结果表明:两者相互作用形成稳定的复合物,最大吸收波长为509nm,与2,7-二氯荧光素的最大吸收波长502nm比较,红移了7nm;两者之间主要通过静电引力结合,但并不排除疏水作用力和氢键;两者之间的结合数为32.     

Nonextensive Statistical Mechanics Application to Vibrational Dynamics of Protein Folding

The vibrational dynamics of protein folding is analyzed in the framework of Tsallis statistics. We employ exact expressions for classical harmonic oscillators by considering the unnormalized constraints. As q→1, we show that these approximations agree with the result of Gaussian network model.     

On-line cysteine modification for protein analysis: new probes for electrochemical tagging nanospray mass spectrometry

A series of electrogenerated selective electrophiles based on substituted benzoquinones has been characterized as tags for l-cysteine and cysteine residues in proteins. The electrophiles are generated electrochemically from the corresponding hydroquinones. It is shown from mass spectrometry analysis that the electrogenerated benzoquinone can tag the biomolecules. The rate constants pertaining to the addition of l-cysteine onto the electrogenerated benzoquinones have been determined using electrochemical techniques. The substitution patterns have been unraveled leading to the assessment of site-specific rate constants. It is shown that the rate constants are primarily dependent on the electronic nature of the substituents as expressed by the Hammett substitution constant. The apparent tagging yields observed for l-cysteine in nanospray mass spectrometry experiments do not correspond to the yields expected from the electrochemical study, as the ionisation efficiencies are highly dependent on the tag. Finally, the on-line tagging has been tested using β-lactoglobulin A and myoglobin. Based on these results, it is concluded that the tagging reaction is selective towards cysteine when it takes place in the nanospray interface. The results show that the methodology presented can be used for a rapid characterization and identification of reactive sites in biomolecules.     

A Novel Model for Protein Immobilization on Microspheres

The immobilization of proteins, especially receptor proteins commonly used in high through-put screening of drugs (HTS), have received great attention in recent years. There are many successful isothermal models for describing the adsorption of protein onto solid surface, such as Langmuir model, Bi-Langmuir model, Fowler model, Freundlich model, Freundlich-Langmuir model and Tekmin model etc. In all these models, Langmuir model was the most favorable one model accepted by many researchers, but the experimental results showed that it was not entirely fit to all adsorption behaviors. So new models were required for describing protein adsorption onto microspheres in different conditions.In our research, a novel isothermal model, including Langmuir and other adsorbing behaviors was presented basing on the holding degree of surface active sites and the interaction styles of protein immobilization. In Langmuir model, the adsorbing amount of protein was described as [PS] =Km[P]/1 + K[P], where [PS] was the concentration of adsorbed protein, [P] was the concentration of freeprotein at equilibrium state, and Km and K was constant. According to the interactions of protein and ligands, there were three patterns in the interactions of protein and ligands. On the similar assumption that the interaction of protein and microspheres were three styles, and based on the definition of the holding degree of surface active sites (Y), three adsorption behaviors could be described as Y K[ P ]φ/ K[P]φ+1 or ln K + φ ln[P] =ln(Y/1-Y) in which [P] was the concentration of free protein at equilibrium state, and φ and K was constant. Different scale of φ presented different adsorption behaviors, especially when φ was 1, the adsorption behavior was Langmuir adsorbing model. Figure I indicated the different adsorbing results in different adsorption behaviors (φ＞1, φ＜1,and φ=1).