灌喂氯化汞大鼠组织中汞结合金属硫蛋白的色谱/质谱联用表征研究

通过测定大鼠脏器组织中汞的总量、金属硫蛋白(MTs)含量及利用尺寸排阻色谱(SEC)与电感耦合等离子体质谱(ICP-MS)联用技术,分析了灌喂HgCl2大鼠脏器组织中汞与MTs的积累量及金属与MTs的结合形态。结果表明,灌喂HgCl2大鼠各脏器中汞的积累量显著高于其相应的对照组,特别是在肾、肝和睾丸中汞的含量较高,表明此3个脏器受汞的危害最大。MTs的含量水平说明当大鼠脏器受汞污染时,肌体中的MTs将被大量诱导产生以对重金属进行解毒。通过SEC-ICP-MS联用技术获得了组织中的金属与MTs的结合形态,其     

金属硫蛋白异构体及亚型异构体的液相色谱分离与质谱鉴别

建立了金属硫蛋白(MT)异构体及亚型异构体的色谱分离与质谱鉴别方法。将金属硫蛋白混合物通过弱阴离子DEAE Sephadex A-25离子交换柱,结合离线电感耦合等离子体质谱(ICP-MS)对锌诱导金属硫蛋白的两个异构体MT-1和MT-2进行分离和检测;利用Sephadex G-25凝胶排阻色谱柱对得到的两个金属硫蛋白异构体进行脱盐;探索脱盐后的金属硫蛋白异构体在不同色谱条件下的C18反相色谱柱上的保留行为,进而实现各个亚型异构体的分离;通过在线电喷雾质谱检测实现了对金属硫蛋白各个亚型异构体的鉴别。结果表明,通过优化色谱条件,由离子交换色谱及凝胶排阻色谱得到的金属硫蛋白各亚型异构体在酸性条件下均得到了良好的分离,质谱检测结果与前人的文献报道结果一致。该方法可使金属硫蛋白各异构体均达到最佳的分离效果。     

同位素示踪SEC-ICP-MS研究大鼠体内镉的形态

通过建立凝胶排阻色谱分离紫外与电感耦合等离子体质谱在线检测的联用技术,研究了灌喂低浓度镉富集同位素后大鼠各器官中镉与蛋白结合的形态.结果发现,镉主要在肝、肾、睾丸中与硫蛋白结合形成金属硫蛋白.与传统方法相比.该法危害性小、具有更接近慢性中毒、能将重金属来源分为天然来源和人为引入等特点.     

金属硫蛋白的色谱/电喷雾电离质谱联用表征方法

通过反相液相色谱(RPLC)与电喷雾电离质谱(ESI-MS)的联用技术,对镉诱导金属硫蛋白标准物质MT-1和MT-2的结构进行表征分析。采用Vydac C8 反相色谱柱(250 mm×2.1 mm i.d., 5 μm, 30 nm),流动相A为pH 6.0的5 mmol/L乙酸铵水溶液,流动相B为pH 6.0的5 mmol/L乙酸铵的甲醇-水(体积比为1∶1)溶液,流动相流速为0.20 mL/min,在40 min内流动相B的体积分数从10%增加到37.5%进行梯度洗脱。分别用紫外(UV)和ESI-M     

金属硫蛋白MT-2a与Cd(Ⅱ)和Cu(Ⅰ)络合的电喷雾质谱表征

采用电喷雾质谱法(ESI-MS)研究金属硫蛋白(MT,存在α和β两种结构域)-2a与金属离子Cd和Cu的络合作用.MT-2a由反相色谱分离纯化制得,并以电喷雾质谱鉴别.通过ESI-MS考察MT-2a与不同量的Cd和Cu的络合,结果表明,Cd能够优先与MT的α结构域络合,形成M2+4S11结构,并且该络合过程存在着明显的协同效应;Cd与MT的β结构域的络合形式为M2+4S9,其呈现出明显的随机松散络合特性;Cu优先与MT的β结构域络合,其络合形式由Cu4逐渐过渡到更高结合态;在Cu含量较高的条件下,Cu与MT呈现出多种络合方式.     

六氯合铂酸钾与金属硫蛋白的体外反应

本文报道了K₂PtCl₆与兔肝Zn₇MT-Ⅱ和apoMT-Ⅱ的反应包含一个氧化还原反应和一个取代反应。通过紫外可见光谱、园二向色谱、柱层析和X-光电子能谱研究了该反应的性质、铂在反应产物中的键合位置和氧化态。金属硫蛋白(MT)被氧化成单体、双聚和多聚产物，其中含有分子间和分子内CyS-SCy二硫键。Pt(Ⅳ)被还原成Pt(Ⅱ)然后键合于产物中。随着K₂PtCl₆与MT的反应摩尔比和反应时间的增加，键合于产物中的Pt(Ⅱ)的计量数增加而蛋白中所含Zn(Ⅱ)的量减少。当Zn₇MT与4和超过10摩尔的K₂PtCl₆反应时，分别得到了Pt₄Zn₄MT和Pt₈MT。当apoMT与7及超过25倍的K₂PtCl₆在pH 2条件下反应时，分别得到了Pt₇MT和Pt₁₅MT。动力学数据表明K₂PtCl₆与apoMT的反应比与Zn₇MT的反应快。     

高效液相色谱-电感耦合等离子体质谱联用测定生物样品中无机汞和甲基汞

建立了高效液相色谱(HPLC)和电感耦合等离子体质谱(ICP-MS)联用测定多种生物样品中的无机汞和甲基汞的方法,并对比了提取生物样品中无机汞和甲基汞的不同前处理方法。实验使用5 mol/L的盐酸超声波提取样品中的无机汞和甲基汞。高效液相色谱流动相为含有0.06 mol/L醋酸氨,20μg/L B i,0.1%(V/V)2-巯基乙醇的5%(V/V)甲醇-水溶液,色谱柱为C18反相柱(5μm,3.9 mm×150 mm)。提取液在液相色谱中分离后,进入电感耦合等离子体质谱检测其中无机汞和甲基汞的浓度。测定了人发(GBW 07601),对虾(GBW 08572),鱼肉组织(IAEA MA-B-3/TM)和牛肝(GBW 080193)4种生物标准参考物,结果与标准参考物的标准值相符。无机汞和甲基汞检出限分别为0.3和0.2μg/L。     

朝鲜淫羊藿中黄酮类化合物的高效液相色谱与电喷雾质谱联用研究

利用高效液相色谱与电喷雾质谱联用技术研究了朝鲜淫羊藿中的黄酮类化合物。实验采用反相C18色谱柱，二元线性梯度洗脱，分离并检测了朝鲜淫羊藿中的6种黄酮类化合物；通过与电喷雾质谱联用获得了这几种化合物的分子量信息，利用质谱的源内碰撞诱导解离技术对这几种化合物进行了结构鉴定。     

兔肝金属硫蛋白亚型异构体的高效液相色谱分离与ESI-MS,MALDI-TOF-MS鉴定

由于金属硫蛋白(MT)基因的多态性,决定不同亚型的MT异构体的存在,MT亚型异构体的结构是MT功能研究的基础.通过离子交换柱可将MT分成MT-1和MT-2两个异构体,用不同条件的反相高效液相色谱(RP-HPLC)可将MT-1和MT-2分成不同的亚型异构体,并利用MALDI-TOFMS和LC-ESI-MS对比确定了它们的分子量.结果表明,兔肝MT在不同的pH条件下分离得到不同分子量的亚型异构体.在酸性条件下,MT-1可分为2个主要亚型异构体,分子量分别为6149.0和6244.5,而MT-2主要分为3个亚型异构体,分子量分别为6149.0,6244.0和6127.0.MT-1和MT-2有2个亚型异构体分子量相同的异构体存在.在酸性条件下,MT-1的2个异构体及MT-2分子量为6127的亚型异构体可稳定存在.     

溶胶-凝胶伴随相分离制备SiO2多孔块体

以正硅酸甲酯(TMOS)为前驱体, 0.01 mol·L^-1盐酸(HCl)为催化剂, 环氧丙烷(PO)为凝胶促进剂, 粘均分子量(M_v)为10000的聚氧化乙烯(PEO)为相分离诱导剂, 采用溶胶-凝胶伴随相分离制备SiO₂多孔块体材料,利用差热分析(DTA)、傅里叶变换红外(FT-IR)光谱、扫描电镜(SEM)、X射线衍射(XRD)、汞压、N₂吸附/脱附等测试技术对所制得的SiO₂多孔块体进行了表征, 探讨了环氧化物调控溶胶-凝胶以及PEO诱导相分离机理. 结果表明, 加入PEO能诱导SiO₂凝胶发生相分离, 当PEO/TMOS摩尔比为0.0018时, 可以获得共连续多孔结构的SiO₂块体材料, 其大孔孔径分布在1-3 μm之间, 比表面积达719 m²·g^-1, 孔体积为0.48 m³·g^-1. 环氧丙烷因其环氧原子的强亲核性和不可逆的开环反应, 促进溶胶-凝胶转换, 同时借助吸附在SiO₂低聚物上的PEO诱导SiO₂凝胶相分离, 从而制备共连续大孔及骨架结构的多孔块体.     

Investigation of metal-binding metallothioneins in the tissues of rats after oral intake of cinnabar

The multi-metal-binding MT fractions in rat tissues after oral intake of cinnabar were characterized by hyphenated size-exclusion chromatography (SEC) and inductively coupled plasma mass spectrometry (ICP–MS). With the increase of both the feeding time and dosage of cinnabar the amounts of Hg-binding MT fractions in rat kidney of groups fed cinnabar increased significantly compared with the control group. Meanwhile, more Cu-binding MT were synthesized in the rat kidney, which confirmed the manipulating effect of MT in the homeostasis of Cu for detoxification of nephritic mercury. Although the Hg-binding MT fractions in rat liver of all cinnabar groups were almost independent of cinnabar dosage (2.5–5.0 g kg^–1 bw) and feeding time (2–4 weeks), the amount was much higher than that of the control group. The amounts of Hg-MT in other rat tissues of the cinnabar groups and the control group were compared and their significance is discussed.     

Investigation of mercury metallothionein complexes in tissues of rat after oral intake of HgCl2

Mercury, a highly toxic metal found widely throughout the environment, is a potent inducer of metallothionein (MT) expression. The role of MTs in the detoxification of mercury after its oral intake in mammals is studied. After feeding rats with mercuric chloride by gastric gavage, the distribution of heavy metals in rat tissues was investigated by inductively coupled plasma mass spectrometry (ICP‐MS). Extensive accumulation of mercury, copper and zinc in kidney and liver is observed. A homemade preparative size‐exclusion chromatography (SEC) column (30 cm × 1.9 cm) packed with Sephadex G‐75 (40–120 µm particle size) gel (Pharmacia) was used for the purification of MT fractions in rat tissues. Preliminary results from SEC indicate that the mercury‐binding MT levels in liver were much lower than in kidney. The MT fractions were collected, desalted, and then separated by reversed‐phase high‐performance liquid chromatography (HPLC) with UV–Vis spectrometry, ICP‐MS and electrospray ionization MS detection. One major and several minor peaks were observed in the HPLC chromatograms of the MT fraction for the kidney sample. UV absorption spectra indicate that MTs were found to bind with mercury. There were no significant mercury‐binding MTs detected in the liver sample using UV detection. ICP‐MS detection showed that mercury‐binding MTs in kidney contained large amounts of mercury and copper but little zinc. Further characterization with ESI‐MS showed that the major peak found in kidney contained Hg₆Cu, Hg₅Cu₂‐MT‐2c and Hg₆‐MT‐2β, Hg₆Cu‐MT‐1γ, Hg₇‐MT‐2α. However, distinction between copper and zinc could not be made based on current mass spectrometric analysis because of instrumental resolution limitations. Copyright © 2005 John Wiley & Sons, Ltd.     

Comparison of different setups for one- and two-dimensional capillary high-performance liquid chromatography and pressurized capillary electrochromatography coupled on-line with mass spectrometry

A comparison of different separation methods (high-performance liquid chromatography (HPLC), capillary HPLC (CHPLC) and pressurized capillary electrochromatography (pCEC)) coupled on-line with mass spectrometry (MS) is undertaken using the separation of a crude extract of ergot fungus (secalis cornuti) as an example. New and simple setups for a two-dimensional CHPLC coupled on-line with electrospray ionization (ESI)-MS (2D-CHPLC-MS) as well as for capillary size-exclusion chromatography performed under pCEC conditions and coupled on-line with ESI-MS (CSEC-pCEC-MS) are shown. In addition, an improved method for column packing is presented.     

Analytical protocol for investigation of zinc speciation in plant tissue

A specific procedure is proposed for investigating the chemical speciation of zinc (Zn) in plant tissues, viz., the extraction of Zn compounds from Plantago lanceolata L. followed by the chromatographic separation and inductively coupled plasma mass spectrometry (ICP-MS) identification of these compounds. In order to separate the Zn compounds, both size-exclusion (SEC) and ionexchange liquid chromatography (IC) were used in direct sequential and reverse sequential modes. In the direct sequential mode, the entire extract undergoes SEC separation and then the individual fractions are injected onto the ion-exchange column. The molecular size distribution is evaluated by SEC coupled on-line to the UV detector. In the reverse sequential mode, the entire extract undergoes the ion-exchange chromatographic separation and then the individual fractions are injected onto the size-exclusion column. The identification of Zn incorporated into the compounds is further performed using ICP-MS. This procedure is particularly useful in speciation studies when identification of the individual components of the element is problematic due to the lack of suitable standard substances, as is the case for Zn compounds. The proposed procedure facilitates assignment of the signals to the individual components of the fractions for both types of chromatography, thus rendering the chemical speciation of Zn possible when the lack of suitable standard substances impedes the identification of individual components.     

Multi-element speciation of metalloproteins in fish tissue using size-exclusion chromatography coupled "on-line" with ICP-isotope dilution-time-of-flight-mass spectrometry

An on-line isotope dilution (ID) method in combination with the coupling of size-exclusion high-performance liquid chromatography (SE-HPLC) to an axial inductively coupled plasma time-of-flight mass spectrometer (ICP-TOFMS) was applied to the quantitative speciation of Cu, Zn and Cd in carp and eel cytosols. Quantitative information on the distribution of essential and toxic metals between the different molecular weight fractions in kidney cytosols of control and Cd exposed carp was obtained with species-unspecific spiking by post-column addition of the enriched isotopes 65Cu, 67Zn and 106Cd. The isotope ratios 63Cu/65Cu, 64Zn/67Zn and 114Cd/106Cd, were monitored by ICP-TOFMS in transient signal mode. The determination of the total cytosolic element concentration was performed by flow injection conventional nebulization ICP-TOFMS. Speciation differences were encountered between control and Cd-stressed carp, indicating that quantitative speciation studies might be useful for ecotoxicological and biomonitoring purposes. Application of this methodology to quantitation of metals associated with metallothioneins (MT) in European eel, sampled at three different sites in Flanders, Belgium was also addressed. A significant increase of the amount of Cd, Cu and Zn bound to hepatic MT was observed with the increase of the total metal and MT concentrations of hepatic eel tissue.     

A shotgun tandem mass spectrometric analysis of phospholipids with normal-phase and/or reverse-phase liquid chromatography/electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry is used in lipidomics studies. The present research established a top-down liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) shotgun analysis method for phospholipids (PLs) using a normal-phase column or a C30 reverse-phase column with the data-dependent MS/MS scanning mode. A normal-phase column can separate most of the major different classes of PLs. By using LC/ESI-MS/MS with a normal-phase column, approximately 50 molecular species were identified in a PL mixture from rat liver. When the reverse-phase column was used, the PLs could be separated depending on their hydrophobicity, essentially the length of their fatty acyl chains and the number of unsaturated bonds in them. The LC/ESI-MS/MS method using a C30 reverse-phase column was applied to phosphatidylcholine (PC) and phosphatidylethanolamine (PE) mixtures as test samples. Molecular species with the same molecular mass but with different pairs of fatty acyl chains were separately identified. As a result, about 60 PC and 50 PE species were identified. PLs from rat liver were subjected to LC/ESI-MS/MS using the C30 reverse-phase column and about 110 molecular species were identified. Off-line two-dimensional LC/ESI-MS/MS with the normal-phase and C30 reverse-phase columns allowed more accurate identification of molecular species by using one-dimensional C30 reverse-phase LC/ESI-MS/MS analysis of the collected fractions.