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.     

纳升级电喷雾离子源-四极杆-飞行时间质谱中磷酸化肽的离子抑制作用

目前磷酸化蛋白质组学研究中的主要技术是蛋白质酶解产生的磷酸化肽的质谱检测。但是实际样品中的磷酸化肽(特别是多磷酸化肽)很难被检测到。其原因普遍认为是由于质谱检测时,非磷酸化肽抑制磷酸化肽。但也有认为非磷酸化肽对磷酸化肽没有抑制作用。另外磷酸化肽之间是否存在离子抑制作用还没有报道。本文采用相同氨基酸序列的标准磷酸化肽和非磷酸化肽,将其单独和混合进行质谱检测,通过对比混合前后磷酸化肽的信号强度,证明了非磷酸化肽对磷酸化肽有离子抑制作用;单磷酸化肽对二磷酸化肽有一定的抑制作用,但不太显著;单磷酸化肽对三磷酸化肽、二磷酸化肽对三磷酸化肽均有显著的离子抑制作用。该研究为今后单磷酸化肽和多磷酸化肽的分段富集和检测提供了有力的证明。     

亲水模式下流动相pH值对磷酸化肽在Click OEG-CD材料上富集选择性的影响

以标准蛋白质α-酪蛋白的酶解液作为研究对象,考察流动相pH值对磷酸化肽在Click OEG-CD材料上富集选择性的影响。首先以磷酸苯二钠作为模型化合物考察流动相pH值对其在Click OEG-CD材料上的保留影响,结果表明当pH值低于磷酸根的pK_a值时,磷酸苯二钠难以电离,与材料的离子交换作用较弱,因而保留也较弱。然后在亲水模式下流动相pH值分别为2, 4, 6时考察Click OEG-CD材料对α-酪蛋白的酶解液中磷酸化肽的富集选择性影响。结果表明,当流动相pH为2时,磷酸化肽不能被材料富集;当pH为4时,磷酸化肽能够被富集,而且洗脱窗口较窄;当pH为6时,磷酸化肽也能够被富集,但是洗脱窗口较宽。因此适合亲水模式下富集磷酸化肽的流动相pH值为4。本研究结果能够为今后将Click OEG-CD材料更好的应用于磷酸化肽富集提供有意义的借鉴。     

微柱液相色谱-电喷雾串联质谱检测多磷酸化肽位点

基于碱性磷酸酶的去磷酸化作用,发展了一种可改善多磷酸化肽电喷雾质谱检测效果的技术。将β-酪蛋白酶解产物用TiO2柱富集后,用碱性磷酸酶进行处理,并经微柱液相色谱分离后采用串联质谱进行鉴定。通过谱图中存在相对分子质量与根据氨基酸序列预计的单磷酸化肽相差80 的色谱峰,可以证实样品中含有单磷酸化肽。此外,经碱性磷酸酶处理后的样品的色谱峰数目的增加,说明样品中可能存在多磷酸化肽段。通过控制去磷酸化反应的程度,使四磷酸化肽的部分磷酸基团被去除,从而可以推断其中3 个磷酸化位点可能处于氨基酸残基序列的第17、18和19位。     

磷酸化肽段分离富集方法研究进展

目前磷酸化肽段鉴定主要依赖于质谱技术,但磷酸化肽段的低丰度性以及来自非磷酸化肽段的干扰等因素,影响质谱的分析与鉴定。因此质谱分析前磷酸化肽段的富集,是深入研究磷酸化蛋白质组学的先决条件。该文介绍了磷酸化蛋白质组学中传统的以及新建立的一些磷酸化肽段分离富集方法的原理及优缺点,这些方法包括固相金属离子亲和色谱法(IMAC)、金属氧化亲和色谱法(MOAC)、强阳/阴离子交换色谱法(SCX/SAX)、亲水相互作用色谱法(HILIC)、静电排斥亲水相互作用色谱法(ERLIC)、化学衍生法、MALDI靶盘富集法以及多种富集方法相结合。     

12种剧毒鹅膏菌的肽类毒素成分鉴定及其相对含量差异比较研究

采用高效液相色谱-高分辨质谱联用技术对12种剧毒鹅膏菌的肽类毒素成分及其相对含量进行了比较研究.样品经含0.5%甲酸-50%甲醇溶液提取, 采用Agilent 300Extend-C18色谱柱, 含0.05% TFA的20 mmol/L NH4Ac溶液-甲醇为流动相, 梯度洗脱分离.12种鹅膏菌的高效液相色谱-质谱联用分析结果表明, 各鹅膏菌所含毒素种类及相对含量差异较大, 呈现显著的色谱指纹特征信息, 12种鹅膏菌中共检测出19种化合物, 13种属于已知鹅膏肽类毒素, 5种为未知鹅膏肽类毒素, 1种为未知小分子化合物组分.本研究为准确识别鹅膏菌的种类、鹅膏肽类毒素的鉴定、预防和鉴别鹅膏毒肽中毒, 提供了科学依据.     

合成受体与模型磷酸化肽的相互作用机理

为了研究基于Zn2+-二甲基吡啶胺及胍羰基吡咯基团的配位型受体Zn Dpa G与磷酸化肽的相互作用机制,选取具有不同序列的磷酸化肽作用模型,采用等温滴定微量热法考察了Zn Dpa G与磷酸化肽的结合常数,研究了模型肽中磷酸基团的数量、密度及位置等因素对多肽与受体间结合强度的影响.结果表明,Zn Dpa G受体对双磷酸化肽结合能力显著高于单磷酸化肽,其结合常数可提高10~40倍,2个磷酸基团的距离越近,结合作用越强;而磷酸基团的位置显著影响受体与单磷酸化肽的结合强度.本研究结果为进一步优化磷酸化肽受体结构设计,实现肽与受体间高选择性识别提供了一定的理论依据.     

高效液相色谱-串联质谱法测定动物肝脏中20种全氟烷基类化合物

建立了Qu ECh ERs-高效液相色谱-串联质谱(HPLC-MS/MS)同时测定动物肝脏中20种全氟烷基类化合物(Perfluorinated alkyl substances,PFAS)残留量的分析方法。样品用0.1%HCl-乙腈振荡提取,C18、N-丙基乙二胺(PSA)和石墨化碳黑(GCB)净化,在C18色谱柱,以5 mmol/L NH4Ac甲醇溶液和5 mmol/L NH4Ac溶液为流动相进行梯度洗脱。多反应监测(MRM)负离子模式,采用基质匹配同位素内标法和外标法结合进行定量分析。20种PFAS在0.1~10μg/L浓度范围内呈线性关系,相关系数不小于0.995;检出限为0.05~0.2μg/kg,定量限为0.4~0.5μg/kg。动物肝脏中20种PFAS的3个浓度水平(0.5,2和5μg/kg)加标的平均回收率为70.3%~108.1%,相对标准偏差(RSD)在2.1%~11.9%(n=6)之间。     

质谱法分析大肠癌组织中踝蛋白的磷酸化修饰

踝蛋白的磷酸化修饰,特别是肿瘤等病理条件下的踝蛋白磷酸化状态,与肿瘤的发病、转移机理密切相关。本研究采用盐析、离子交换层析和电泳分离并纯化了人大肠癌组织中的踝蛋白(Talin),经胰酶水解获得其肽段混合物,进一步分别利用固定化Fe3+亲和层析和TiO2亲和层析在酸性条件下对其中磷酸化修饰肽段进行吸附,并以1%氨水进行洗脱。在Michrom Magic C18色谱柱上,以A:99%水+1%乙腈+0.1%甲酸和B:99%乙腈+1%水+0.1%甲酸两种流动相进行梯度洗脱分离,采用ESI质谱进行依赖数据的二级子离子扫描。结果显示,固定化Fe3+富集到8个磷酸化肽段而TiO2富集到9个磷酸化肽段。本研究提供了一种快速、准确地鉴定从人大肠癌组织中分离表征踝蛋白的方法。     

介孔氧化铁材料快速、高效富集分离磷酸化肽段和蛋白的新方法研究

以0.1μmol/Lβ-casein磷酸化蛋白酶解液为对象,利用在酸性条件下对PO43-能够特异性吸附的氧化铁材料为新载体,对介孔氧化铁材料富集分离磷酸化肽段的孵育液酸含量、孵育液有机溶剂含量和洗脱液选择的条件进行了优化,结果表明:室温条件下,在含有0.1%乙酸和30%乙腈的孵育液中孵育5min后,经1mol/LNH3.H2O溶液的洗脱,介孔氧化铁可有效地将磷酸化肽段从蛋白酶解液中富集分离。本方法也可以选择性地提取α-casein磷酸化蛋白,实现了简单、快速、高效的磷酸化肽段和蛋白的富集分离。同时,通过MALDI-TOF串级质谱分析,成功地完成了在优化条件下分离出的磷酸化肽段磷酸位点的鉴定。     

非那雄胺合成工艺改进

非那雄胺能抑制5α-还原酶的活性,明显降低二氢睾酮水平,是一种治疗良性前列腺增生的有效药品。该合成工艺以甾烯酮酸为原料,将其与氯化亚砜反应,无须分离即与叔丁胺反应得17β-酰胺化合物,再氧化开环,环合,氢化,脱氢合成了非那雄胺。经元素分析、IR、1HNMR、13CNMR、MS分析表征了其结构。该法无须使用昂贵的2,2-二吡啶二硫化物和剧毒药品苯亚硒酸酐,且以乙酸铵代替氨气,降低了对设备的要求和腐蚀,更适用于工业生产。     

Dichroism of absorption and polarization of phosphorescence of molecules of the closed form of nitrobenzospirothiopyran

The spectral-polarization characteristics of absorption and phosphorescence of molecules of the initial form of nitro-substituted indolinospirobenzothiopyran were studied in oriented polyethylene films and in solutions with different polarity. An oscillator model of the electron transitions responsible for the formation of absorption and luminescence spectra was suggested. It was established that the principal differences in the spectral and photophysical properties of the compound studied and its oxygen-containing analog are associated with the fact that the electronegativity of the S atom is lower than that of the O atom. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 6, pp. 1143–1146, June, 1997.     

Synthesis of phenolic components of Grains of Paradise

Two vanilloids, (5E)-8-(4-hydroxy-3-methoxyphenyl)oct-5-en-4-one (1) and 4-[3-hydroxydecyl]-2-methoxyphenol (2), isolated from the dried seeds of Grains of Paradise (Aframomum melegueta), were synthesized; the latter compound was made as the S-enantiomer and the material derived from the seeds was found to be a 1:1.7 mixture of the R and S isomers. The synthetic route used should allow the preparation of analogs having extended alkyl chains and consequently different lipophilicity, and 3, a homolog of 2, was also prepared.     

Acid hydrolysis of carboxymethylcellulose of low degree of substitution

Cotton cellulose was swollen in a sodium hydroxide solution and carboxymethylated by a two-bath method for different periods of time for each process. The kinetics of acid hydrolysis and the crystallinity of the swollen and carboxymethylated samples were measured. The proportion of broken bonds, rate constants for hydrolysis, and permeability of cellulose to hydrolyzing agents were calculated. The susceptibility of glycosidic linkages to acid hydrolysis was improved by carboxymethylation more than by swelling in alkali. The increased accessibility of carboxymethylcellulose to acid was regarded as a consequence of increased intra-and intercrystalline swelling and of the glycosidic bonds' weakness caused by the electron-attracting carboxymethyl group on the C-6 position.     

Study of hydration of two cements of different strengths

Main hydration products of two cement pastes, i.e. CSH-gel, portlandite (P) (and specific surface S) were studied by static heating, and by SEM, TEM and XRD, as a function of cement strength (C-33 and C-43) hydration time (th) and subsequent hydration in water vapour.Total change in mass on hydration and air drying, M_o, increased with strength of cement paste and with hydration time. Content of water escaping at 110 to 220°C, defined as water bound with low energy, mainly interlayer and hydrate water, was independent on cement strength but its content increased with (th). Content of chemically bound (zeolitic) water in CSH-gel, escaping at 220-400°C, was slightly dependent on strength and increased with (th). It was possibly derived from the dehydroxylation of CSH-gel and AFm phase. Portlandite water, escaping at 400-500°C, was independent on cement strength and was higher on longer hydration. Large P crystals were formed in the weaker cement paste C-33. Smaller crystals were formed in C-43 but they increased with (th). Carbonate formated on contact with air (calcite, vaterite and aragonite), decomposed in cement at 600-700^oC. It was high in pastes C-33(1 month) and C-43(1 month), i.e. 5.7 and 3.3%, respectively; it was less than 1% after 6 hydration months (low sensitivity to carbonation) in agreement with the XRD study showing carbonates in the air dry paste (1month), and its absence on prolonged hydration (6 months) and on acetone treatment. Water vapour treatment of (6 months) pastes or wetting-drying increased this sensitivity.Nanosized P-crystals, detected by TEM, could contribute to the cement strength; carbonate was observed on the rims of gel clusters.This revised version was published online in November 2005 with corrections to the Cover Date.     

恶臭嗅辨实验室建设探讨

针对恶臭测试的环境影响问题,提出了解决的实例方案,并对方案的要点及优缺点进行讨论,此方案在实际操作中具有较好的效果。     

Estimation of Kinetic Parameters of Thermal Oxidation of Ilmenite

The aim of the presented work was the investigation of thermal oxidation of ilmenite in static air atmosphere. The investigations were carried out by use of a derivatograph (MOM, Hungary). The changes of crystallographic structure of investigated samples were identified by X-ray diffractometry on Philips PW-1710 diffractometer. In temperature above 500°C appears structure of hematite Fe₂O₃. On the basis of the thermogravimetric measurements, the contracting area and contracting volume models were found as the best fitting experimental data. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mechanism of action of base-catalyzed oxygenation of phenol derivatives

Cyclopropyl derivative of 2,6-di-tert-butylphenol is synthesized as a probe to investigate the mechanism of base-catalyzed autooxidation of phenol derivatives. Our study indicates that one electron reduction of molecular oxygen from phenolate gives phenoxyl radical 3, a key intermediate of autooxidation. The coupling of phenoxyl radical and superoxide radical gives peroxylate anion 4 and produces the final epoxy alcohol adduct 6.     

氯氧化镁生成热的研究

用氧化镁氯化镁水溶液制备了8水合氯氧化镁[nMg(OH)2·MgCl2·8H2O], 并测定了其在盐酸中的溶解热, 实验结果表明, 氯氧化镁溶解热与n值呈线性关系, 根据溶解热求出5Mg(OH)2·MgCl2·8H2O和3Mg(OH)2·MgCl2·8H2O的生成热分别为-7727.1和5888.1kJ·mol^1^-。