用同步辐射X-荧光定量测定电泳分离后蛋白条带内的微量元素

建立了同步辐射X荧-光(SRXRF)定量测定生物样品等电聚焦(IEF)分离后蛋白条带内的微量元素Fe、Cu和Zn的方法。用薄层聚丙烯酰胺凝胶分离人血红蛋白后,用SRXRF测定了各亚型条带内的金属含量,用加一定量金属的含蛋白聚丙烯酰胺凝胶做SRXRF定量测定蛋白条带内微量元素Fe、Cu和Zn的定量标准,校准曲线线性回归系数r在0~8μg/g范围内均大于0.99;检出限分别为2.43、1.12和0.96μg/g;测定蛋白条带内Fe和Zn的回收率分别为90.4%和115.7%。该联用技术可用于生物样品中微量元素的化学形态分析,同时给出蛋白质的微量元素组成和等电点等信息。     

堆肥过程不同分子量水溶性有机物电子转移能力的演变及影响因素

利用超滤技术、电化学方法和光谱技术, 以堆肥水溶性有机物的不同分子量(MW)组分为研究对象, 分析在堆肥过程中不同分子量水溶性有机物(DOM)的组成特征、结构演变和电子转移能力变化的影响因素.结果表明, 类蛋白物质主要存在于堆肥前期的DOM(MW<1 kDa)中, 随着堆肥的进行, 类蛋白物质不断降解, 类富里酸物质持续合成, 堆肥后期类蛋白物质被完全降解, 类富里酸物质成为DOM(MW<1 kDa)主要的荧光组分.类腐殖物质是DOM(MW=1～3 kDa)、DOM(MW=3～5 kDa)和DOM(MW>5 kDa)的主要荧光组分, 堆肥过程中类腐殖质物质在3种不同分子量组分的变化各不相同, 但是堆肥后期类腐殖质物质在3个不同分子量组分的含量均高于堆肥初期. 堆肥过程中DOM(MW<1 kDa)的电子供给能力(EDC)呈降低趋势, 而电子接受能力(EAC)呈升高趋势; DOM(MW>5 kDa)的EDC在堆肥过程中呈上升趋势, 而EAC则无明显的变化规律.DOM(MW=1～3 kDa)和DOM(MW=3～5 kDa)的EDC和EAC在整个堆肥过程无明显变化规律.不同分子量组分堆肥DOM 的EAC受控于堆肥过程木质素降解产物的含量, 而其EDC变化与荧光参数和紫外参数无明显关系.     

体积排阻色谱-电感耦合等离子体质谱分析富硒大米含硒蛋白组成

建立体积排阻色谱-电感耦合等离子体质谱(SEC-HPLC-ICP-MS)联用技术分析富硒大米含硒蛋白组成方法。通过水提、盐提、碱提和醇提方法提取,并用丙酮沉淀蛋白,硒的回收率分别为9.6%,16.8%,48.2%和14.9%,纯化后的蛋白结合硒的量由大到小依次为碱溶谷蛋白>球蛋白>醇溶蛋白>清蛋白。蛋白液经SEC-HPLC-ICP-MS检测,通过蛋白色谱峰(λ=280 nm)和ICP-MS硒峰(78Se)对比分析,利用分子量标准曲线测定出4类蛋白中含硒蛋白的分子量。结果表明,富硒大米中清蛋白和醇溶蛋白并不是硒的主要存在蛋白。硒主要存在于>7 kDa的碱溶谷蛋白和球蛋白,其中碱溶含硒蛋白主要组分F1分子量为199.8 kDa。     

人胎盘膜铁蛋白同类型双亚基功能、铁核结构和释放铁动力学研究

以人胎盘组织为实验材料,小批量制备电泳纯人胎盘膜铁蛋白(HPMF),对其结构与功能进行研究.十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)技术揭示,HPMF蛋白壳由分子量分别为15 kDa(MF15)和20 kDa(MF20)的亚基组成,其中MF15蛋白含量约为MF20的3倍.经肽质量指纹图谱(PMF)技术鉴定...     

不同分子尺寸溶解性有机物的光解行为研究

研究了三个分子量区间(0.45μm~100kDa,100~5kDa和5kDa)的溶解性有机物(DOM)在不同光源辐照下的光解行为。溶解有机碳(DOC)和UV254的测试结果表明,光解可以有效地减少所有分子量区间的DOM总量,而且UVC光源的存在可以进一步促进DOM的降解。三维荧光光谱测定结合平行因子分析,识别出3个腐殖质类荧光组分(C1:UVA+UVC,C2:UVA+UVB,C3:UVC)。在光解过程中,较大分子腐殖质类(5kDa)的荧光强度相对较稳定,甚至增强;而小分子腐殖质类(5kDa)的荧光组分C1和C2具有显著的光解行为,UVC的存在可以促进两种荧光组分的光解。所有分子量区间的C3组分都发生了光生成现象。     

多壁碳纳米管对质谱分析中的血清蛋白富集作用研究

通过多壁碳纳米管(MWCNTs)对临床血清蛋白提取物进行富集处理,经表面增强激光解析离子化飞行时间质谱(SELDI-TOF-MS)检测,发现MWCNTs对血清中小分子量蛋白(<20 kDa)具有很好的富集效果。同时还考察了内径、长度等参数对血清蛋白富集效果的影响。该方法可用于临床血清样本中低丰度的小分子量蛋白的检测。     

微量元素对围产儿影响的研究

为了解信宜市微量元素(碘、锌、铁、铅)对围产儿的影响，对1871例围产儿脐血进行了检测，并对早产儿、小于胎龄儿与健康足月适于胎龄儿作比较。结果表明，四种微量元素失衡的发生率分别为：早产儿缺锌23．63％，缺碘0．90％，缺铁0．90％，铅高43．63％；小于胎龄儿缺锌25．30％，缺碘1．20％，缺铁1．20％，铅高40．96%；与正常组(缺锌11．32％，缺碘1．72％，缺铁1．19％，铅高20．02％)相比，血锌均明显低于正常组，血铅均明显高于对照组，血碘、铁均无明显差异。早产并小于胎龄儿在缺锌、高铅状态下死亡率最高(占小于胎龄儿50％)。提示缺锌、铅高状态对围产儿发育有重要影响，必须加强围产儿保健，及时诊治微量元素失衡。     

肌肉组织中四环素类抗生素的固相萃取-液相色谱法测定

建立了四环素类抗生素的高灵敏度测定方法，通过对实验条件的优化，采用固相萃取-反相高效液相色谱法同时测定肌肉组织中的土霉素(oxytetracycline)、四环素(tetracycline)、金霉素(chlortetracycline)3种四环素类抗生素残留量；土霉素、四环素、金霉素的检出限分别为20、40、100μg／kg；方法相对标准偏差为5．1％-7．1％(n＝5)，平均回收率为80％—82％。     

双孢菇同工凝集素的制备及性质研究

本文提出了双孢菇凝集素（ＡＢＡ）的制备及性质研究，采用稀盐溶液浸提、酒精分级沉淀、ＤＥ－２３和ＣＭ－２３柱层析分离纯化，得Ａ、Ｂ两大组分，经ＰＡＧ检测组分Ａ为三条带，组分Ｂ为两条带。ＳＤＳ－ＰＡＧＥ测定表观分子量：组分Ａ三条还分别为８２０００、６１０００、５１０００；组分Ｂ两条带分别为２３０００、２２０００。由ＩＦＥ测得等电点：Ａ组分ＰＩ分别为ＰＨ５．０６、ＰＨ５．４６、ＰＨ６．０１；组分Ｂ等电占     

聚乳酸/聚氧化乙烯共混体系的热行为和力学性能及流变行为

采用熔融共混方法制备了聚乳酸与聚氧化乙烯的共混物.细致研究了重均分子量分别为2 kDa、10kDa1、00 kDa和600 kDa的聚氧化乙烯对聚乳酸的改性效果,并使用DSC、DMA及旋转流变仪等分析了共混物的相容性、热行为、力学性能和流变行为.结果表明,在聚氧化乙烯的组分含量不超过20 wt%的前提下,共混体系保持为完全相容体系,当聚氧化乙烯的分子量超过10 kDa时,其对聚乳酸的增塑效果,不随分子量增加而降低;增加聚氧化乙烯的分子量,可以提高材料的弹性模量和熔体强度.     

Detection of metalloproteins in human liver cytosol by synchrotron radiation X-ray fluorescence combined with gel filtration chromatography and isoelectric focusing separation

Synchrotron radiation X-ray fluorescence (SRXRF) spectroscopy is an advanced method of quantitative multielemental analysis with space resolution of several microm and sensitivities in the microg g(-1) range. It can be used for keeping track of trace elements after an electrophoretic separation of biological samples. In this paper, proteins in human liver cytosol were separated with gel filtration chromatography and thin layer isoelectric focusing (IEF). The contents of metal ions in protein bands were determined by SRXRF. The results showed that in the molecular weight (MW) range of 10 approximately 25 kDa, there were at least 2 Zn-containing bands with isoelectric point (pI) of 5 approximately 6 and 6.2 approximately 7, respectively and about 11 Fe-containing proteins with pI of 4.4, 4.6, 4.8, 5.0, 5.2, 5.3, 5.5, 5.6, 6.6, 6.8, and 7.2, respectively, present in human liver cytosol. The Zn-containing band with pI of 5-6 is the dominant species of zinc in this MW range. In addition, the Cu-containing bands with pI of 5.0 and below 4.8 were also detected. It is demonstrated that the procedure could be widely used in further investigations of the chemical species of trace elements in biological samples.     

Change of concentrations of trace elements and protein contents in the liver of zinc deficient mice

The concentrations of essential trace elements and proteins in cytosolic fraction of hepatic cells of mice fed with Zn-deficient diet (Zn-def, mice) and control were determined by ICP-MS and BCA protein assay method, respectively, after division into forty fractions by gel filtration chromatography. The concentrations of zinc and proteins decreased in the 14–17th fractions of Zn-def, mice, whereas cobalt concentrations increased in the 14, 17, and 18th fractions. However, no significant differences were found on the gel after SDS-PAGE for the 12–21st fractions, although the BCA protein assay date showed the decrease of protein amounts in 13–15th fractions of Zn-def. mice.     

X-ray fluorescence spectrometry analysis for minerals with agaron gel for sample preparation

A mineral sample preparation with agaron gel used in X-ray fluorescence analysis was developed. Mineral samples were decomposed with aqua-regia. The sample solution mixed with agaron gel was heated to boiling and then it would become quasi-solid gel under normal temperature. The elements dispersed in gel medium could be detected by X-ray fluorescence spectrometry. The method has both advantages of the solution preparation method and the solid preparation method. In addition, this method was adapted in detecting high content ores due to its' avoiding the risk of using platinum crucibles. The method has been applied to the determination of lead, zinc and iron in zinc and lead concentrates successfully.     

Analysis of metal-containing proteins by gel electrophoresis and synchrotron radiation X-ray fluorescence

For the analysis of metal-containing proteins, sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been combined with synchrotron radiation X-ray fluorescence (SRXRF). In a pilot study the applicability of this combined method was tested in the analysis of metalloaded apoazurin and of the selenoproteins in rat testis homogenate. It was shown that it can be applied in the determination of the major stable binding forms of trace elements. After further improvement of the limits of detection the method will allow the analysis of trace element-containing proteins present in the samples at low concentrations. This revised version was published online in August 2006 with corrections to the Cover Date.     

Optimizing total reflection X-ray fluorescence for direct trace element quantification in proteins I: Influence of sample homogeneity and reflector type

Total reflection X-ray fluorescence (TXRF) is a very promising method for the direct, quick and reliable multi-elemental quantification of trace elements in protein samples. With the introduction of an internal standard consisting of two reference elements, scandium and gallium, a wide range of proteins can be analyzed, regardless of their salt content, buffer composition, additives and amino acid composition. This strategy also enables quantification of matrix effects. Two potential issues associated with drying have been considered in this study: (1) Formation of heterogeneous residues of varying thickness and/or density; and (2) separation of the internal standard and protein during drying (which has to be prevented to allow accurate quantification). These issues were investigated by microbeam X-ray fluorescence (μXRF) with special emphasis on (I) the influence of sample support and (II) the protein / buffer system used. In the first part, a model protein was studied on well established sample supports used in TXRF, PIXE and XRF (Mylar, siliconized quartz, Plexiglas and silicon). In the second part we imaged proteins of different molecular weight, oligomerization state, bound metals and solubility.     

Speciation of metal ions in proteins by combining PIXE and thin layer electrophoresis

Particle induced X-ray emission (PIXE) spectroscopy is a simple and convenient method of quantitative multielemental analysis with sensitivities in the μg/g range, that can be successfully used for trace analysis of metal ions in proteins or enzymes. However, due to its elemental character the technique alone is not a priori suitable for speciation. Keeping track of the metal ions of interest throughout a proper biochemical separation technique, on the other hand, could be a useful strategy for speciation. Different versions of thin layer electrophoresis (polyacrylamide gel, agarose or cellulose acetate electrophoresis) are very effective and sensitive methods to separate proteins or protein fragments. Due to the high absolute sensitivity of PIXE the metal ions concentrated in the narrow bands of an electropherogram can be in situ successfully detected. The present paper describes this unique combination of biochemical separation and ion beam analysis which significantly extends the information obtained from electrophoresis. Illustrative applications are given and the advantages and limitations of the method are discussed. Possible extensions of the technique are also outlined.     

轻度铅中毒对儿童微量元素及骨龄的影响

为探讨儿童轻度铅中毒对微量元素及骨龄的影响,采用钨舟原子化法和原子吸收光谱法分别检测了33例轻度铅中毒患儿血铅及微量元素水平,用X摄片进行骨龄测定,并进行了相关分析。结果表明,轻度铅中毒患儿微量元素钙、铁、锌降低,骨龄发育落后,血铅含量与钙、铁、锌水平呈负相关。提示轻度铅中毒使儿童骨龄落后,可能与微量元素间的失衡有关。     

尺寸排阻-反相液相色谱-质谱联用技术在大鼠肾脏翻译后修饰蛋白质鉴定中的应用

LC-MS联用技术在蛋白质组学研究中具有重要的作用,但是在复杂的生物体系中,由于样品的高度复杂性和其中蛋白质含量的巨大差异,执行全面且无倾向的蛋白质组分析是一项挑战。因此,在液相色谱分离中采用基于不同原理的色谱分离方法来降低蛋白质样本的复杂度,并对微量蛋白质进行富集,对后续采用质谱方法进行信息的采集和深入分析至关重要。在这里我们开发了一种基于尺寸排阻色谱(SEC)与反相液相色谱(RPLC)结合的新方法来进行复杂体系蛋白质的分离和鉴定,特别是对于微量蛋白质的分析。首先使用SEC对蛋白质进行分离和富集,并酶解成多肽,再通过RPLC-MS联用的方法对酶解后的多肽进行分离和鉴定。结果显示使用上述方法可以有效降低蛋白质样本的复杂度,并有效提高微量蛋白质的鉴定能力,可从大鼠肾脏鉴定出23621个肽段及1345个蛋白质,比常规的二维强阳离子交换-反相液相色谱法(2D SCX-RPLC)鉴定到的肽段及蛋白质分别多出69%及27%。此外,该方法对肾脏翻译后修饰(PTM)蛋白质的鉴定显示出更多的优势,翻译后修饰的多肽鉴定率显著增加,特别是磷酸化肽段的鉴定效率可达到靶向富集策略的水平。在此展示的SEC-RPLC-MS可以更好地了解蛋白质翻译后修饰对肾脏的影响,最终将有助于增加我们对正常的生理性肾功能以及病理过程机制的理解。     

Determination of ppb concentrations of transition metals by radioisotope-excited energy-dispersive X-ray spectrometry

Using ammonium pyrrolidine dithiocarbamate as precipitant and iron as a carrier, ppb concentrations of the following elements were coprecipitated from fresh water: vanadium, chromium, manganese, nickel, copper, zinc, selenium, mercury, and lead. Precipitates were collected on membrane filters then element concentrations were determined using energy dispersive X-ray fluorescence spectrometry. Calibration curves indicate sensitivities ranging from 0.4 ppb for V, Zn, As and Hg to 1.2 ppb for Pb. It is likely that this method of preconcentration can be directly incorporated into field sampling procedures, thus eliminating the problems of sample contamination or trace element losses by absorption on container walls.     

Sample Preparation and X-Ray Fluorescence Analysis of Sulfide Ores

Sulfide ores with varied concentrations, including four ore reference materials (zinc, copper, and iron sulfides) and two copper-nickel and iron sulfide samples, were used to develop an improved method for their analysis by X-ray fluorescence. The optimum conditions were established for the fusion of homogeneous stable glass disks. Different weights of silica were added to the samples, which were then fused with a lithium borate flux. The amount of silica varied from 1:20 for samples with low sulfur concentrations to 1:5 for those with high concentrations. The weight ratio of sample (including silica) to lithium tetraborate was 1:14. Lithium nitrate was used as a pre-oxidizing reagent to prevent degradation of platinum–gold crucibles. After pre-oxidation at 580°C for 13 minutes, the temperature was increased to 1100°C for 12 minutes. The relative standard deviations for major and trace elements were generally better than 5% and 10%, respectively.