Digestion method for silver accumulated in micro-organisms

Silver is accumulated to high concentrations in certain microbial strains. Here a bomb digestion method is proposed, using HNO₃ and HCl, for the extraction and digestion of silver and silver compounds from the organic matrix. The method is applicable for the quantitative determination of silver by inductively coupled plasma atomic emission spectroscopy.     

微波消解-电感耦合等离子体发射光谱测定铜精矿中的银

铜精矿试样利用HCl和HNO3混酸于微波消解仪中进行溶解,电感耦合等离子体发射光谱(ICP-OES)法测定铜精矿中的银含量。试验了不同比例消解体系的消解效果,研究了不同浓度铁、铜基体的影响,结果表明王水体系消解效果最佳,1 000mg/L以下的铁、铜或铁、铜的混合基体对银的测定没有影响,10%HCl体系作为测定酸度。该方法对银的线性范围为0.73~1 500g/t,标准物质测定值与标准值无显著性差异。该方法前处理试剂消耗少、速度快,分析浓度范围宽,可用于铜精矿中银的快速测定。     

微波密闭消解-ICP-MS－标准加入法测定矿石中金和银

建立了微波密闭消解-电感耦合等离子体质谱法(ICP-MS)采用标准加入法测定矿石中痕量元素金和银。考察了微波试样消解、基体效应、质谱干扰,并进行了ICP离子源以及质谱仪检测条件和微波消解参数的最优化。以标准加入法消除复杂多变的矿石基体对分析信号的影响,干扰校正方程消除多原子离子等质谱重叠影响。本法测定矿石中金银结果表明:回收率为197Au 106%～113%、107Ag 95%～105%、 109Ag 93%～103%,相对标准偏差RSD <6%（n=8）,检测限197Au为10 ng/g、107Ag为3 ng/g、109Ag为6 ng/g。方法适用性强,可满足不同类型矿石中超痕量金银的测定,分析步骤少,操作简便,快捷准确     

电感耦合等离子体光谱法(ICP-AES)测定银精矿中铜、铅、锌、砷、镉、钙、镁、锰含量

针对银精矿样品复杂,难消解的特点,研究了不同酸溶法和碱熔法对样品的消解情况,建立了硝酸,盐酸,氢氟酸,高氯酸消解银精矿的方法。根据元素灵敏度和抗干扰性,选定各元素的测定波长。通过酸溶样和碱熔样测定结果比对,验证了方法准确性。建立了四酸消解-电感耦合等离子体光谱法测定银精矿中铜、铅、锌、砷、镉、钙、镁、锰含量的方法,元素的线性相关系数均在0.9999以上。通过共存元素干扰实验,确定了银精矿中高含量元素(铜、铅、锌、铁、锑、铋等)对测定元素结果没有影响。方法检出限：Cu 0.0063 mg/L, Pb 0.0159 mg/L ,Zn 0.0090 mg/L,As 0.0192 mg/L, Cd 0.0093 mg/L ,Ca 0.0084 mg/L, Mg 0.0075 mg/L, Mn 0.0081 mg/L。测定下限：Cu 0.0105mg/L,Pb 0.0265 mg/L, Zn 0.0150 mg/L, As 0.0320 mg/L, Cd 0.0155 mg/L, Ca 0.0140 mg/L, Mg 0.0125 mg/L,Mn 0.0135 mg/L。3个样品的相对标准偏差在0.87%~3.56%之间,加标回收率在95.00%~103.56%之间。方法流程短,操作简单,快速,灵敏度和再现性高,结果准确可靠,可以满足银精矿中铜、铅、锌、砷、镉、钙、镁、锰含量的测定。     

Peptide mapping of polypeptides separated by two-dimensional electrophoresis: protease digestion directly on the two-dimensional gel followed by electrophoresis in reverse direction

A method is described which allows to reveal simultaneously the proteolytic patterns of numerous polypeptides separated by two-dimensional electrophoresis. After two-dimensional electrophoresis, the gels were dipped successively in buffers for preequilibration, protease digestion, and reequilibration. They were then returned to the electrophoresis tank, and electrophoresis was continued for a short time. After silver staining, digestion products appeared, lined up behind the original polypeptide spots. The method allows proteolytic patterns of numerous polypeptides to be visualized simply and quickly. Among proteins of wheat leaves, 31 groups of related polypeptides were found according to the similarity of their proteolytic patterns.     

Silver stain removal using H2O2 for enhanced peptide mass mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Hydrogen peroxide solutions are reported for the removal of silver stain from proteins isolated in polyacrylamide gels. Removal of silver stain prior to in-gel digestion is shown to enhance sensitivity and sequence coverage of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) peptide mass maps. The rate of silver removal using H2O2 is influenced by H2O2 concentration and increases with increasing pH. The presence of complexation reagents such as ammonia from mass spectrometry compatible ammonium bicarbonate solutions enhances the efficiency and speed of H2O2-mediated silver removal. H2O2-mediated silver removal using the described procedure does not appear to have any detrimental effects on proteins but is observed to produce a slightly elevated level of methionine oxidization over that usually observed in in-gel tryptic digestion.     

微波消解-ICP-OES法测定鲢鱼肌肉中的19种矿物元素

以硝酸和过氧化氢混合溶液为消解溶剂,利用微波消解法制备鲢鱼肌肉样品溶液,再应用全谱直读电感耦合等离子体发射光谱测定技术(ICP-OES),对鲢鱼肌肉中所含的矿物元素进行较为全面的定性定量分析。定性结果显示,鲢鱼肌肉中含有19种矿物元素,分别是K,Ti,Al,Ga,B,Ba,Ca,Cd,Cr,Cu,Fe,Mg,Mn,Na,P,Pb,S,Sr,Zn等。定量结果显示,ICP-OES定量分析线性范围宽,可达2个数量级以上;工作曲线线性相关系数在0.999以上;样品测定RSD值在0.44 % ~ 11.83 %之间,大部分在5%以内;除了一个元素以外,回收率测定结果都在(100±10)%以内;测定结果可为相关应用研究提供参考数据。     

A statistical comparison of silver and SYPRO Ruby staining for proteomic analysis

Silver staining has been the method most commonly employed for high sensitivity staining of proteins following two-dimensional gel electrophoresis. Whilst this method offers detection in the nanogram range it does have major drawbacks including a lack of linearity, nonstoichiometric staining of proteins, a lack of compatibility with the microchemical preparation of proteins for identification by mass spectrometric techniques, and a highly subjective assessment of the staining endpoint. SYPRO Ruby is a relatively new, ruthenium complex-based stain which is reported to offer advantages over silver, particularly in overcoming the limitations cited above. We describe a series of experiments where several protein staining procedures commonly employed are compared. To enable optimization of the in situ digestion procedure, a statistical approach has been undertaken. The effects of a variety of staining, digestion, and analysis protocols on the downstream processing of a test radiolabeled protein were studied. The data confirms that as well as offering sensitivity similar to silver, SYPRO Ruby staining is reproducible, linear, and offers a higher level of compatibility with the identification of proteins by mass spectrometry.     

快速测定CODcr的研究

对以往经典的测定水样ＣＯＤｃｒ的方法进行了改进，研究了消解时间与酸度的关系，确定了最佳条件，并确定了用硝酸银沉淀氯离子，消除氯离子的干扰取有毒的硫酸汞是可行的。     

An optimized procedure for detection of proteins on carrier ampholyte isoelectric focusing and immobilized pH gradient gels with imidazole and zinc salts: its application to the identification of isoelectric focusing separated isoforms by in-gel proteolysis and mass spectrometry analysis.

A method for the characterization of proteins separated by isoelectric focusing in carrier ampholytes (CA-IEF) or immobilized pH gradient (IPG) gels by in-gel digestion and mass spectrometry is described. Proteins are detected by an improved imidazole-Sodium dodecyl sulfate (SDS)-zinc staining adapted for IEF and IPG gels. Sensitivity is close to that of mass spectrometry-compatible silver staining, but simpler and faster. Proteins were digested in imidazole-SDS-zinc stained CA-IEF and IPG gels in the presence of a zinc-chelating agent. Mass spectra were clearly interpretable as carrier ampholytes which were efficiently removed before digestion; high-sequence coverage that allowed isoform characterization was obtained by analyzing both the aqueous and the organic phase extracts.     

Detection and Determination of Released Ions in the Presence of Nanoparticles: Selectivity or Strategy?

Metallic nanoparticles can release ionic species, but also both species can occur in the same samples. Therefore, there is a need of efficient and cost‐effective methods to determine these ionic species in the presence of the corresponding nanoparticles. Electroanalytical techniques open the door to this selective detection of NPs and their ions. In this work, a methodology that allows the direct determination of ionic silver (Ag⁺) in the presence of silver nanoparticles based on anodic stripping voltammetry was implemented. Silver nanoparticles were determined, after acidic digestion of the sample, by difference with respect to the total content of silver. The method was validated in terms of specific identification of silver ions, linearity, working range, limit of detection, limit of quantification, recovery, repeatability and ruggedness. All parameters are adequate for an analytical method following Eurachem recommendations. The validated method was used to determine the concentration of Ag⁺ and total silver in two commercial products of colloidal silver. The results were compared with those obtained by atomic absorption spectrometry in combination with an ultrafiltration step for isolation of ionic silver. There were no significant differences in the results. The proposed methodology benefits from the intrinsic selectivity of the electroanalysis methods, allowing to eliminate the steps of pretreatments of the samples, which are necessary in other techniques. The novelty of the article lies in the direct determination of Ag (I) ions in the presence of AgNPs, without the use of previous separation steps.     

Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

High-sensitivity, high-throughput analysis of proteins for proteomics studies is usually performed by polyacrylamide gel electrophoresis in combination with mass spectrometry. However, the quality of the data obtained depends on the in-gel digestion procedure employed. This work describes an improvement in the in-gel digestion efficiency for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis. A dramatic improvement in the coverage of tryptic peptides was observed when n-octyl glucoside was added to the buffer. Whole cell extracted proteins from S. cerevisiae were separated by two-dimensional gel electrophoresis and stained with silver. Protein spots were identified using our improved in-gel digestion method and MALDI-TOFMS. In addition, the mass spectra obtained by using the matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) were compared with those obtained using 2,5-dihydroxybenzoic acid (DHB). The DHB matrix usually gave more peaks, which led to higher sequence coverage and, consequently, to higher confidence in protein identification. This improved in-gel digestion protocol is simple and useful for protein identification by MALDI-TOFMS.     

Stabilization of thin-layer agarose gels after isoelectric focusing with polyacrylamide enables reverse imidazole-zinc staining and facilitates two-dimensional gel electrophoresis

Large-pore-size agarose gels provide excellent resolving capacity for high molecular weight biomolecules. Thin-layer agarose isoelectric focusing (IEF) gels on polyester support films are especially useful for the separation of large proteins based on their pI in native conformation, but the method has suffered from the lack of detection methods compatible with agarose gels in hydrated form. Recently, an acrylamide copolymerization method was reported to enable mass-spectrometry-compatible silver staining and in-gel digestion of proteins. In this study, the method was further applied by demonstrating successful reverse imidazole-zinc staining of thin-layer agarose IEF gels copolymerized with acrylamide. The sensitivity of the reverse staining method on the composite gel at its best equaled the sensitivity of the traditional dried agarose silver staining method. Owing to the increased durability and reversible detection, the reverse-stained first-dimension gel could be conveniently prepared for the second-dimension sodium dodecyl sulfate polyacrylamide gel electrophoresis by reduction and alkylation. In addition, the micropreparative generation of tryptic peptides of Coomassie brilliant blue R-250 stained proteins in the composite gel is demonstrated.     

Comparison of different digestion methods for total decomposition of siliceous and organic environmental samples.

A comparison was made of different digestion methods for the total decomposition of siliceous and organically environmental samples prior to their analysis by inductively coupled plasma optical emission spectrometry (ICP-OES). In the present study, three different digestion methods, including microwave-assisted, hot plate heating and pressurized digestion (pressure bomb), were employed for the determination of nine heavy metals, i.e. Ag, Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn in sediment, soil, sludge and oil. The investigation of different combinations of acids through their analytical performance demonstrated that HCl plays a vital role in the determination of silver. The combination of HNO3 and HCl possesses more reactive ability in oxidizing organic matter. The recoveries of all elements of interest in sediment (NIST 2704) obtained by different digestion methods were found to be 86% to 113%, while microwave assisted digestion with various combinations of HNO3-HCl-HF and HNO3-HClO4-HF was considered to be a viable alternative to the conventional digestion systems because of its more intensive reaction conditions. The analytical results of four certified reference materials with different matrices, including sediment (GBW 07305), soil (GBW 07411), sludge (BCR R-143) and oil (NIST 1085a), by the microwave-assisted acid digestion method indicated that the recoveries of all elements of interest were more than 85% and the throughput of applied analytical method could be elevated significantly.     

电感耦合等离子体质谱法测定地球化学样品中的银、砷、锑、铋

建立了王水分解样品,电感耦合等离子体质谱法测定地球化学样品中的银、砷、锑、铋的方法,采用王水-水浴分解试样,电感耦合等离子体质谱仪测定新疆和田策勒恰哈地球化学样品中的银、砷、锑、铋,确立了最佳的溶样时间、测定酸度、干扰元素的校正方法,结果令人满意。各元素的方法检出限为0.003 8~0.041g/g;相对标准偏差RSD(n=12)为0.52%~2.8%;方法灵敏度较好、简便、快速,且结果准确,适用于大批量地球化学样品的快速测定。     

微波密闭消解-电感耦合等离子体质谱法(ICP-MS)测定矿石中金和银

建立了微波密闭消解-电感耦合等离子体质谱法(ICP-MS)测定矿石中痕量元素金和银。考察了微波消解试样、基体效应、质谱干扰,并进行了ICP离子源以及质谱仪检测条件和微波消解参数的最优化。以标准加入法消除复杂多变的矿石基体对分析信号的影响,干扰校正方程消除多原子离子等质谱重叠影响。测定矿石中金银结果表明:回收率为197Au106%～113%、107Ag95%～105%、109Ag93%～103%,相对标准偏差RSD<6%(n=8),检测限197Au、107Ag、109Ag分别为10、3、6ng/g。方法适用性强,可满足不同类型矿石中超痕量金银的测定,分析步骤少,操作简便,快捷准确。     

小分子表面活性剂在胶内酶切增效中的应用

本研究成功地将一种有机小分子表面活性剂RapiGest SF(Waters)用于改进电泳分离的蛋白质的鉴定效率。通过基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)的肽质量指纹谱,考察了酶切时间、加入量、加样次序、点靶方法对方法灵敏度、蛋白质鉴定率的影响。RapiGest SF浓度为0.5%~1%,在酶切之前加入可获得更多的肽段峰和更高的鉴定率。本方法考染体系的总灵敏度为332fmol,银染体系为664fmol。比较了RapiGest SF与MALDI-TOF-MS和电喷雾质谱(ESI-MS)兼容性,未观察到明显的负影响。方法操作简便,重复性较好,适合鉴定电泳分离的低丰度蛋白质。     

Ultrasound-assisted extraction in the determination of arsenic,cadmium, copper,lead, and silver in contaminated soil samples by inductively coupled plasma atomic emission spectrometry

An extraction method was developed for the determination of toxic elements in contaminated soil samples by inductively coupled plasma atomic emission spectrometry (ICP-AES). The determination of arsenic, cadmium, lead, and silver in ultrasound-assisted extracts of SRM 2710 and SRM 2711 by ICP-AES was carried out with high accuracy and precision (RSD<3.7%). The certified concentrations of the SRMs were obtained for arsenic, cadmium, lead, and silver by using an ultrasound-assisted extraction method with a digestion solution of (1+1)-diluted aqua regia. The determination of copper in SRMs by the ultrasound-assisted extraction method and analysis by ICP-AES failed to obtain the certified concentrations at the 95% level of confidence using (+/-2 s) as confidence limits of the mean. However, the same results were observed with the use of the microwave digestion method and reflux, which is the ISO 11466 standard method. The analysis of the SRMs showed that the ultrasound-assisted extraction method is highly comparable with the other methods used for such purposes. The major advantages of the ultrasound-assisted extraction method compared to the microwave and reflux methods are the high treatment rate (50 samples simultaneously in nine minutes) and low reagent usage, the main benefit of which are the low chloride and nitrate concentrations in the extracts.     

Improved technique for recovery and measurement of polonium-210 from environmental materials

Polonium-210 obtained in acidic solution by digestion of sediment or tissue samples is efficiently plated onto one side of a silver disc for measurement of α-activity. Optimum conditions for solutions obtained from environmental samples are pH 1.5–2 in the presence of citrate and ascorbic acid, with the solution at 80°C and the silver disc rotated at 100–150 radian s^?1. Observed recoveries using a polonium-208 tracer are 97.5 ± 2.5% for sediments and 99.3 ± 1.3% for tissues.     

原子转移自由基聚合修饰银丝固定化酶反应器的制备及在蛋白质组鉴定中的应用

针对传统溶液酶解存在的酶解时间较长、酶自切物干扰以及蛋白酶不能重复使用等缺陷,通过电子转移生成催化剂的原子转移自由基聚合法修饰银丝,并以其为载体制备了一种新型的固定化酶反应器。用质谱考察了银丝固定化酶反应器(SW-Trypsin)的酶解效率、重复性和回收率。结果表明:绒毛状聚合物修饰的SW-Trypsin的酶解效率较高,酶解标准蛋白牛血清白蛋白(BSA)20 min后,肽段的氨基酸序列覆盖率可达93%,高于传统溶液酶解方法酶解16 h所得79%的覆盖率。使用该固定化酶反应器于一个月内8次酶解BSA所得的氨基酸序列覆盖率在89%到97%之间,平均覆盖率为94%,显示出良好的稳定性。另外,该固定化酶反应器酶解马心肌红蛋白(MYO)的回收率为87.67%。最后,用SW-Trypsin酶解腾冲嗜热菌全蛋白20 min,所鉴定到的氨基酸序列覆盖率和蛋白数量与同样条件下溶液酶解16 h的结果接近,且零漏切位点肽段的比例更高。加之容易分离的优点,SW-Trypsin在蛋白质组学的应用中具有良好的前景。