Absolute quantification of superoxide dismutase in cytosol and mitochondria of mice hepatic cells exposed to mercury by a novel metallomic approach

In the last years, the development of new methods for analyzing accurate and precise individual metalloproteins is of increasing importance, since numerous metalloproteins are excellent biomarkers of oxidative stress and diseases. In that way, methods based on the use of post column isotopic dilution analysis (IDA) or enriched protein standards are required to obtain a sufficient degree of accuracy, precision and high limits of detection. This paper reports the identification and absolute quantification of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in cytosol and mitochondria from mice hepatic cells using a innovative column switching analytical approach. The method consisted of orthogonal chromatographic systems coupled to inductively coupling plasma-mass spectrometry equipped with a octopole reaction systems (ICP-ORS-MS) and UV detectors: size exclusion fractionation (SEC) of the cytosolic and mitochondrial extracts followed by online anion exchange chromatographic (AEC) separation of Cu/Zn containing species. After purification, Cu,Zn-SOD was identified after tryptic digestion by molecular mass spectrometry (MS). The MS/MS spectrum of a doubly charged peptide was used to obtain the sequence of the protein using the MASCOT searching engine. This optimized methodology reduces the time of analysis and avoids the use of sample preconcentration and clean-up procedures, such as cut-off centrifuged filters, solid phase extraction (SPE), precipitation procedures, off-line fractions insolates, etc. In this sense, the method is robust, reliable and fast with typical chromatographic run time less than 20 min. Precision in terms of relative standard deviation (n = 5) is of 3–5% and detection limits is 0.21 ng Cu g⁻¹.     

Determination of δ‐[L‐α‐aminoadipyl]‐L‐cysteinyl‐D‐valine in cell extracts of Penicillium chrysogenum using ion pair‐RP‐UPLC‐MS/MS

δ‐[L ‐α‐Aminoadipyl]‐L ‐cysteinyl‐D ‐valine (ACV) is a key intermediate in the biosynthesis pathway of penicillins and cephalosporins. Therefore, the accurate quantification of ACV is relevant, e.g. for kinetic studies on the production of these β‐lactam antibiotics. However, accurate quantification of ACV is a challenge, because it is an active thiol compound which, upon exposure to air, can easily react with other thiol compounds to form oxidized disulfides. We have found that, during exposure to air, the oxidation of ACV occurs both in aqueous standard solutions as well as in biological samples. Qualitative and quantitative determinations of ACV and the oxidized dimer bis‐δ‐[L ‐α‐aminoadipyl]‐L ‐cysteinyl‐D ‐valine have been carried out using ion pair reversed‐phase ultra high‐performance liquid chromatography, hyphenated with tandem mass spectrometry (IP‐RP‐UPLC‐MS/MS) as the analytical platform. We show that by application of tris(2‐carboxy‐ethyl)phosphine hydrochloride (TCEP) as the reducing reagent, the total amount of ACV can be determined, while using maleimide as derivatizing reagent enables to quantify the free reduced form only.     

蛋白质中二硫键的定位及其质谱分析*

二硫键是一种常见的蛋白质翻译后修饰,对稳定蛋白质的空间结构,保持及调节其生物活性等都有着非常重要的作用。因此,确定二硫键在蛋白质中的位置是全面了解含二硫键蛋白化学结构的重要方面。在众多实验方法中,现代质谱技术因其操作简单、快速、灵敏等优点而成为分析二硫键的重要手段。本文介绍了目前主要的定位二硫键的方法,以及质谱在二硫键定位分析中的应用与进展。     

Investigation of protein FTT1103 electroactivity using carbon and mercury electrodes. Surface-inhibition approach for disulfide oxidoreductases using silver amalgam powder

Recently, it was shown that electrochemical methods can be used for analysis of poorly water-soluble proteins and for study of their structural changes and intermolecular (protein–ligand) interactions. In this study, we focused on complex electrochemical investigation of recombinant protein FTT1103, a disulfide oxidoreductase with structural similarity to well described DsbA proteins. This thioredoxin-like periplasmic lipoprotein plays an important role in virulence of bacteria Francisella tularensis. For electrochemical analyses, adsorptive transfer (ex situ) square-wave voltammetry with pyrolytic graphite electrode, and alternating-current voltammetry and constant-current chronopotentiometric stripping analysis with mercury electrodes, including silver solid amalgam electrode (AgSAE) were used. AgSAE was used in poorly water-soluble protein analysis for the first time. In addition to basic redox, electrocatalytic and adsorption/desorption characterization of FTT1103, electrochemical methods were also used for sensitive determination of the protein at nanomolar level and study of its interaction with surface of AgSA microparticles. Proposed electrochemical protocol and AgSA surface-inhibition approach presented here could be used in future for biochemical studies focused on proteins associated with membranes as well as on those with disulfide oxidoreductase activity.     

分散液-液微萃取与气相色谱-质谱联用分析环境水样中痕量的2,4-二硝基甲苯和磷酸三(2-氯乙基)酯

建立了分散液-液微萃取与气相色谱-质谱联用同时测定环境水样中痕量2,4-二硝基甲苯和磷酸三(2-氯乙基)酯的新方法。对影响萃取效率的因素进行了详细的考察和优化,确定采用的最佳萃取条件为: 将0.8 mL乙醇和60 μL氯仿的混合溶液快速注入5.0 mL的样品溶液中,振动混匀120 s后,离心分离,吸取沉积在试管底部的氯仿相直接进样分析。该方法对磷酸三(2-氯乙基)酯和2,4-二硝基甲苯的检出限(信噪比为3)分别为0.01和0.04 μg/L,富集倍数分别为96.6和127.5;两种物质的线性范围达3到4个数量级;日内和日间测定的相对标准偏差(RSDs, n=6)分别为8.6%～11.5%和8.9%～12.0%。将该方法用于环境水样中2,4-二硝基甲苯和磷酸三(2-氯乙基)酯的分析,其加标回收率为102.1%～110.9%。方法具有操作简单、方便快速、灵敏度高、无交叉污染和环境友好等优点。     

磷酸三(2-氯乙基)酯标准物质的研制

研制了有机磷阻燃剂磷酸三(2-氯乙基)酯(TCEP)标准物质。采用硅胶柱层析法对TCEP原料进行纯化,得到纯度大于99%的TCEP纯品。通过红外光谱和气相色谱–质谱准确定性分析后,利用气相色谱法和气相色谱–质谱法结合热重分析法分别对TCEP纯物质进行纯度定值。将制备的50 g TCEP纯品分装到100个小瓶中,每瓶0.5g,从中随机选取10瓶,进行均匀性和稳定性实验,结果表明,研制的TCEP标准物质均匀性良好,在半年内是稳定的。研制的有机磷阻燃剂磷酸三(2-氯乙基)酯标准物质的纯度定值为99.56%,扩展不确定度为1.18%(k=2)。研制的磷酸三(2-氯乙基)酯标准物质满足国家二级标准物质标准的要求。     

混合模式吸附剂固相萃取-气相色谱-质谱法测定皮革中的磷酸三（2-氯乙基）酯

磷酸三（2-氯乙基）酯（TCEP）是一种皮革生产中常用的加工助剂,其具有致癌、神经毒性和生殖毒性,被欧洲化学品管理局列入禁用的第二批授权物质清单。由于皮革产品基质复杂,采用常用的固相萃取（SPE）方法提取对TCEP的回收率不高。Silica-WCX是一种自制的含有羧基与烷基的新型混合模式吸附剂。研究表明,通过在酸性条件下使其羧基保持质子化状态,能有效增强Silica-WCX对极性化合物TCEP的萃取性能,使TCEP的回收率得到明显提高。本文以Silica-WCX为SPE材料,建立了测定皮革中TCEP的SPE-GC-MS方法。该方法的线性范围为0.10~100.0 μg/L,定量限（S/N=10）为44.46 ng/kg,不同添加水平下TCEP的平均回收率在91.45%~99.98%之间,相对标准偏差（RSD）在4.33%~5.97%之间。该方法简便快捷,灵敏度高,定量限低于欧盟《化学品的注册、评估、授权和限制》（registration,evaluation,authorization and restriction of chemicals,REACH）法规的限量要求,适用于皮革及其制品中TCEP的测定。     

Silicon nanowires as field-effect transducers for biosensor development: A review

The unique electronic properties and miniaturized dimensions of silicon nanowires (SiNWs) are attractive for label-free, real-time and sensitive detection of biomolecules. Sensors based on SiNWs operate as field effect transistors (FETs) and can be fabricated either by top–down or bottom–up approaches. Advances in fabrication methods have allowed for the control of physicochemical and electronic properties of SiNWs, providing opportunity for interfacing of SiNW-FET probes with intracellular environments. The Debye screening length is an important consideration that determines the performance and detection limits of SiNW-FET sensors, especially at physiologically relevant conditions of ionic strength (>100 mM). In this review, we discuss the construction and application of SiNW-FET sensors for detection of ions, nucleic acids and protein markers. Advantages and disadvantages of the top–down and bottom–up approaches for synthesis of SiNWs are discussed. An overview of various methods for surface functionalization of SiNWs for immobilization of selective chemistry is provided in the context of impact on the analytical performance of SiNW-FET sensors. In addition to in vitro examples, an overview of the progress of use of SiNW-FET sensors for ex vivo studies is also presented. This review concludes with a discussion of the future prospects of SiNW-FET sensors.     

Synthesis of reversible cyclic peptides

A novel approach for the synthesis of cyclic peptides that can exist in either linear or cyclized conformations is described. Synthesis of the peptides was achieved via a modified solid phase methodology. The reversible linear/cyclized (i.e., open/closed) states are controlled via the reduction/oxidation of a disulfide bond incorporated into the backbone of the peptide chain.