磷酸化蛋白质组学中的分离富集方法研究进展

蛋白质的磷酸化是一种可逆性的翻译后修饰,在细胞的增值、分化、信号转导以及转录与翻译调控、蛋白质复合体的形成、蛋白质降解等方面发挥着极为重要的作用.因此磷酸化蛋白的鉴定成为翻译后修饰研究的重要内容.但由于磷酸化蛋白的丰度较低, 难以用质谱直接检测.为了解决这个问题,改善质谱对磷酸肽的信号响应, 需要对磷酸化蛋白质或磷酸肽进行富集.本文系统地介绍了磷酸化蛋白组学研究中应用较为广泛和最新建立的各种分离富集方法的原理、特点、应用研究进展,包括抗体富集法、激酶特异富集法、亲和富集法、化学修饰法、多种色谱分离富集方法以及MALDI靶盘富集法.     

PCL-b-PDMS-b-PCL复合环氧树脂的表面结构

利用原子力显微镜(AFM)中的敲击模式原子力显微镜(TM-AFM)和摩擦力显微镜(FFM)对不同含量聚己内酯-b-聚二甲基硅氧烷-b-聚己内酯三嵌段(PCL-b-PDMS-b-PCL)共聚物复合环氧树脂的表面富集结构进行了分析研究. TM-AFM测试在不同作用力下得到了PCL-b-PDMS-b-PC 含量不同环氧树脂表面及其亚表面的分相结构; 同时利用FFM对其表面进行摩擦和磨损试验. 结果表明, PCL-b-PDMS-b-PCL含量不同时摩擦性能表现出较大的变化, 当其质量分数达到30%时, 表面性能达到了稳定. 接触角试验也验证了以上的结果.     

浊点萃取光度法测定水样中亚硝酸根

A new method for the determination of trace nitrite by spectrophotometric after cloud point extraction was proposed.The effects of experimental conditions such as acidity,concentration of chromogenic reagent and surfactant,equilibration temperature and time on cloud point extraction were discussed.Under the optimum conditions,a good linear relationship was obtained in the range of 4.0～200 μg/L of the nitrite(r=0.9998),the detection limits of 0.43 μg/L.The recoveries fell in the range from 97.7% to 102.4% an...     

Large volume injection for the direct analysis of ionophores and avermectins in surface water by liquid chromatography-electrospray ionization tandem mass spectrometry

A simple method for the direct determination of residues of ionophore antibiotics and avermectin antiparasitics in surface water is reported. Using a large volume injector, a mixture of methanol and surface water is injected into an analytical liquid chromatography (LC) column and subsequently screened for residues of these veterinary pharmaceuticals using electrospray ionization tandem mass spectrometry. On-line sample enrichment and chromatographic separation are achieved using a single, short (20 mm) octadecylsilane LC column. The method permits the detection of four ionophores and two avermectins in surface water samples at low ng L⁻¹ concentrations. Minimal matrix effects were observed for a variety of surface water samples which permitted the use of reagent-based standards for quantitation.     

Development of core–shell structure Fe3O4@Ta2O5 microspheres for selective enrichment of phosphopeptides for mass spectrometry analysis

Protein phosphorylation is one of the most important post-translational modifications. Due to the dynamic nature and low stoichiometry of the protein phosphorylation, enrichment of phosphopeptides from proteolytic mixtures is often necessary prior to their characterization by mass spectrometry. Many metal oxides such as titanium dioxide and zirconium dioxide have been successfully applied to isolation and enrichment of phosphopeptides. Recently, niobium pentoxide was proved to have the ability for selective enrichment of phosphopeptides. Considering the proximity of tantalum to niobium, we supposed that Ta₂O₅ can be used as affinity probes for phosphopeptide enrichment. In the work, we synthesized Fe₃O₄@Ta₂O₅ magnetic microspheres with core–shell structure for selective enrichment of phosphopeptides. To demonstrate its ability for selective enrichment of phosphopeptides, we applied Fe₃O₄@Ta₂O₅ magnetic microspheres to isolation and enrichment of the phosphopeptides from tryptic digestion of standard proteins and real samples, and then the enriched peptides were analyzed by matrix-assisted laser desorption mass spectrometry analysis (MALDI-MS) or liquid chromatography coupled to electrospray ionization mass spectrometry (LC–ESI-MS). Experiment results demonstrate that Ta₂O₅ coated-magnetic microspheres show the excellent potential for selective enrichment of phosphopeptides.     

污泥堆肥农用中重金属在海桐中的积累效应

采用小区试验的方法,研究了金华污水厂污泥堆肥土地利用过程中海桐根部对重金属的吸收及富集作用。结果表明,海桐对Cu均表现出比Zn更强的富集能力,而对Cd、Pb的富集能力很弱,其须根对重金属的吸收能力显著高于主根;就不同重金属的绝对吸收量而言,对Zn的吸收总量是最高的;须根中的Zn含量总体呈现随土壤中Zn含量增加而上升的趋势。     

Induction and inhibition of preferential enrichment by controlling the mode of the polymorphic transition with seed crystals

Both induction and inhibition of "preferential enrichment", an unusual symmetry-breaking enantiomeric-resolution phenomenon observed upon simple recrystallization of a certain kind of racemic crystals from organic solvents, have been successfully achieved by controlling the mode of the polymorphic transition during crystallization with appropriate seed crystals. Such control of the polymorphic transition can be interpreted in terms of a novel phenomenon consisting of 1) the adsorption of prenucleation aggregates, 2) the heterogeneous nucleation and crystal growth of a metastable crystalline form, and 3) the subsequent polymorphic transition into the more stable form; these three processes occur on the same surface of a seed crystal. We refer to this phenomenon as an "epitaxial transition", which has been confirmed by means of in situ attenuated total reflection (ATR) FTIR spectroscopy in solution and the solid state, differential scanning calorimetry (DSC) measurements of the deposited crystals, and X-ray crystallographic analysis of the single crystals or the direct-space approach employing the Monte Carlo method with the Rietveld refinement for the structure solution from the powder X-ray diffraction data.     

混合模式色谱材料Click TE-GSH高效富集多磷酸化肽

多磷酸蛋白对于生物体适应内外环境具有重要意义,而明确多磷酸蛋白的磷酸位点功能及其信号转导机制尤为关键. 复杂生物样品中多磷酸化肽的低丰度、低电离的特性,以及非磷酸化肽的抑制作用,决定了质谱分析前进行多磷酸化肽富集是非常必要的步骤. 本工作采用基于巯基-烯烃点击化学法合成的混合模式材料Click TE-GSH进行单磷酸化肽和多磷酸化肽的选择性富集. 我们建立了单磷酸化肽、双磷酸化肽和多磷酸化肽的顺序分段富集方法. 该优化方法能抗干扰,应用于脱脂牛奶时富集到11条多磷酸化肽. 与商品化固化金属亲和色谱(IMAC)材料相比,Click TE-GSH富集多磷酸化肽的选择性更好. 本工作所建立的富集方法为高效富集多磷酸化肽提供新方法和新技术.     

在线富集离子色谱-质谱联用法同时测定饲料添加剂中的16种有机酸

建立了在线富集方式结合离子色谱-质谱(IC-MS)快速分离分析16种有机酸的方法。离子色谱配备自制富集柱和分离柱对有机酸进行在线富集和分离;质谱采用大气压化学电离源负离子电离方式(APCI^-),在选择离子监控(SIM)模式下对有机酸进行定性和定量分析。采用200 μL大体积进样,在线富集时间为3 min,以NaOH溶液作为淋洗液,梯度洗脱。结果表明,富集柱和分离柱对有机酸有很好的富集分离能力;16种有机酸在30 min内完全洗脱,并在一定浓度范围内线性关系良好;方法检出限(LODs)为0.01~0.22 mg/L;加标回收率为70.6%~110.8%,相对标准偏差(RSD)≤6.3%。该方法样品前处理简单,分离速度快,有机酸检测灵敏度高,适用于多种饲料添加剂样品中有机酸添加剂的检测。     

聚胍基离子液体材料制备及其对蛋白质识别性能评价

磷酸化修饰是蛋白质翻译后修饰中最为重要的修饰之一,蛋白质的磷酸化修饰几乎参与生命活动的每一个环节。因此,制备对磷酸化蛋白具有选择性识别性能的材料在磷酸化蛋白质组学中具有重要意义。本实验首先合成胍基离子液体功能单体,通过沉淀聚合法合成聚胍基离子液体材料。通过傅里叶红外光谱(FT-IR)、扫描电子显微镜(SEM)、热重分析仪(TGA)考察了材料的结构、形貌、热稳定性。结果显示所制备材料为粒径约200 nm的球形颗粒。并以标准磷酸化蛋白(β-酪蛋白)为模型蛋白质,考察了聚胍基离子液体材料的识别性能。研究结果表明:材料对磷酸化蛋白具有较高吸附容量(对 β-酪蛋白的最大吸附量达到599.1 mg/g)、较快的吸附速度(1 h内达平衡),而且对磷酸化蛋白表现出较高的选择性。