季铵化磁性壳聚糖复合材料对磷酸化肽的富集研究

蛋白质磷酸化是最重要和最普遍的翻译后修饰之一,蛋白质磷酸化的测定对于全面了解生物过程中的磷酸化途径至关重要。质谱技术是分析蛋白质磷酸化的重要手段,但磷酸化肽固有的低丰度、低电离效率以及与非磷酸化肽共存等特性严重影响质谱对其进行直接分析。为解决此问题,需在质谱分析前对磷酸化肽进行选择性富集。本研究制备了一种基于季铵化磁性壳聚糖的复合材料用于磷酸化肽的富集。此磁性材料具有快速的磁响应性、良好的生物相容性、正电性以及廉价易得等优点。采用β-酪蛋白作为模型蛋白质,结果表明此材料对磷酸化肽具有良好的富集选择性。经过富集后,结合基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）检测手段,方法的检出限为0.4 fmol。本方法被成功用于脱脂牛奶中磷酸化肽的检测,表明其对复杂样品中磷酸化肽的富集和检测具有良好的应用潜力。     

聚苯乙烯型双季铵树脂的制备及性能研究

三乙烯二胺和四甲基乙二胺与氯甲基聚苯乙烯反应,得到同时含有季铵和叔胺的强-弱碱型树脂,再用碘甲烷季铵化叔胺,制备聚苯乙烯双季铵树脂;另一种方法是将二甲基苄胺型叔胺树脂用4-溴丁基三甲基溴化铵季铵化引入4碳链双季铵基团。反应结果表明,两类强弱碱基树脂的季铵化反应结果并不理想,二甲基苄胺型叔胺树脂季铵化反应的转化率最高为36%。考察了这些新型双季铵树脂的热稳定性及对水溶液中有机酸(如苯磺酸和对-硝基苯乙酸)的吸附、脱附性能。     

季铵化聚乙烯亚胺的制备

以环氧丙烷为叔胺化试剂,氯化苄为季铵化试剂通过聚乙烯亚胺(PEI)的叔胺化与季铵化反应制备了季铵化的聚乙烯亚胺(QPEI),其结构经IR表征。考察了反应条件对合成QPEI的影响。研究结果表明,使用环氧丙烷,可绿色化地实现PEI链中的伯胺基和仲胺基的叔胺化得到叔胺化的聚乙烯亚胺(TPEI);以氯化苄为季铵化试剂,于50℃反应30 h可使TPEI链中90%以上的叔胺基实现季铵化,从而使PEI链中的总N原子数实现50%以上的季铵化。     

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。     

带有苄基二甲基γ-硅丙基氯化铵侧基的聚硅氧烷的合成

通过γ－氯丙基甲基硅氧烷－二甲基硅氧烷共聚物和Ｎ,Ｎ－二甲基苄基胺的季铵化反应合成了带有苄基二甲基γ－硅丙基氯化铵侧基的聚硅氧烷。通过季铵化反应过程中γ－氯丙基转化程度随时间的变化以及影响该季铵化速率的各种因素的研究,表明该季铵化反应在反应的初期基本遵循二级反应速率定律,提高反应物的浓度、采用与反应物有适当相溶性的高极性溶剂,以及提高反应温度均能加速季铵化反应。     

氯化磷催化下氨基酸的自组装成肽反应

在生命起源与进化研究中,氨基酸的成肽反应一直是人们关注的焦点.我们发现,在无机磷试剂催化下氨基酸可发生自组装反应生成多肽[1,2],曾有报道L-色氨酸与三氯氧磷反应以电喷雾质谱(ESI-MS)检测未观察到肽的形成[3].本文研究了L-甲硫氨酸与五氯化磷、L-脯氨酸与三氯氧磷的成肽反应,并发现了能使L-色氨酸与三氯氧磷反应成肽的方法(Scheme 1).     

季铵化反应的热动力学研究

季铵化反应不仅可作为研究溶剂效应对化学反应性影响的理想对象,也是合成相转移催化剂、表面活性剂和高分子聚电解质的基本方法。研究聚电解质约纶(ionene)的催化性能是模拟酶的途径之一。因此,研究季铵化反应动力学具有重要的理论和实践意义。     

接枝型三相相转移催化剂的制备及其催化作用

将苯乙烯（St）接枝聚合在微米级硅胶表面,制备了接枝微粒PSt／SiO2;使用新型氯甲基化试剂1,4-二氯甲氧基丁烷,对接枝在硅胶表面的聚苯乙烯进行了氯甲基化（CM）反应,制得了氯甲基聚苯乙烯／硅胶（CMPS／SiO2）接枝微粒;使三乙胺与CMPS分子链上的苄氯基团发生季铵化反应,制得了固载有季铵盐（Quaternary salt）的接枝微粒QPSt／SiO2,即制得了接枝型三相相转移催化剂．将此相转移催化剂用于氯化苄与乙酸钠合成乙酸苄酯的相转移催化反应,考察了催化活性、各种因素对相转移催化反应的影响及催化剂的重复使用性能．实验结果表明,接枝型三相相转移催化剂QPSt／SiO2对乙酸苄酯的合成具有较高的催化活性,在液-固-液之间即可有效地实现反应物种乙酸根的转移,在60℃的较低温度下反应7h,氯化苄的转化率可达66．1％;研究发现,固体催化剂QPSt／SiO2表面接枝聚合物PSt的季铵化程度对其催化活性有很大的影响,季铵化程度过大与过小催化活性都较低,当季铵化程度为20％左右时,催化剂的活性最高．     

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。     

分离蛋白质的高效离子交换色谱填料的研究(Ⅳ)──强阴离子交换色谱填料的合成及应用

含环氧基的硅胶与二乙胺或聚乙烯亚胺反应,其产物再与碘甲烷或氯化苄反应,得到含季铵离子的强阴离子交换色谱填料。该填料可用于蛋白质和生物工程产品(如基因重组人干扰素γ、粒细胞-巨噬细胞集落刺激因子和人绒毛膜促性腺素等)的分离纯化。其中,环氧基硅胶与二乙胺加成后再用氯化苄季铵化的产物是较好的强阴离子交换色谱填料。     

Effect of structure of nucleophile and substrate on the quaternization of heterocyclic amines

The influence of the nature of the quaternizing agent and substrate on the quaternization of heterocyclic amines, derivatives of pyridine, β-picoline, nicotinamide, pyridoxine, was studied. The synthesized compounds were characterized by IR spectroscopy and elemental analysis. The conclusions were made about the effect of the structure of nucleophile and substrate on the process of quaternization reaction.     

Development of a derivatisation method for the analysis of aldehyde modified amino acid residues in proteins by Fourier transform mass spectrometry

A method was developed for the analysis of amino acids within bovine serum albumin (BSA) which had been modified by reaction with different enals. BSA was reacted with the aldehydes and the reaction products were stabilised by reaction with NaBH₄. The protein was then hydrolysed with 6N HCl and the hydrolysis products were analysed by liquid chromatography-mass spectrometry (LC-MS). The modified amino acids were derivatised with propylchloroformate. High resolution mass spectrometry carried out using an LTQ-Orbitrap instrument which was able to characterise a wide range of adducts. In addition double adducts were observed to be formed with 4-hydroxynonenal (HNE) and lysine or lysine + histidine. Qualitatively it was possible to consistently observe a pyridinium adduct formed between lysine and pentenal in human plasma from normal subjects.     

Novel synthetic method of phosphonamidate peptides and its application in peptide sequencing via multistage mass spectrometry

Phosphonamidate peptides were prepared in good yields by reaction of ethoxy(phenyl)phosphinate with free peptides in a mixed solution of H2O, C2H5OH, Et3N and CCl4 at room temp.; their in situ electrospray ionization mass spectra exhibited high sensitivity compared with the free peptides, and sequential loss of amino acid residues of the sodiated phosphonamidate peptides from the C-terminus was found in multistage ESI mass spectrometry. The results show that N-phosphonyl derivatization combined with multistage ESI-MS is a powerful method for peptide sequencing.     

O-季铵化-N-壳聚糖Schiff碱的合成及表征

对壳聚糖氨基和羟基进行化学改性,合成了水溶性双功能化壳聚糖衍生物O-季铵化-N-壳聚糖Schiff碱(O-HTCCS);用红外光谱表征了产物的结构;确定了O-HTCCS的最佳合成条件,并测定了其溶解性能.结果表明,合成O-HTCCS的最佳条件为:壳聚糖Schiff碱/缩水甘油三甲基氯化铵(GTMAc)摩尔比为1∶5;反应时间24 h;反应温度70℃.在最佳条件下合成O-HTCCS的产率为78.5%,季铵化度为89.7%.与此同时,O-HTCCS的水溶性随季铵化度的增大而提高,季铵化度达到70%以上能溶于水;且其在有机溶剂中的溶解性优于壳聚糖.     

复方板蓝根颗粒化学成分的质谱研究

利用电喷雾多级串联质谱(ESI-MSn)和傅里叶变换离子回旋共振质谱(FT-ICR-MS)技术,对复方板蓝根颗粒经溶剂萃取后的95%(体积分数)醇提和水提部位的化学成分进行了系统研究,鉴定出多种氨基酸成分、糖及其衍生物、有机酸、氨基酸和单糖的梅拉德反应初级产物以及含硫化合物表告依春.该方法灵敏快速,适宜于中药提取物的化学成分分析.     

Matrix-assisted laser desorption/ionization detection of polymerase chain reaction products by utilizing the 5'-3' exonuclease activity of Thermus aquaticus DNA polymerase

The 5'-3' exonuclease activity of DNA polymerase was utilized in the polymerase chain reaction system to generate a specific signal concomitant with amplification. These signals were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). This method obviates the need to perform extensive DNA purification of reaction products that is often necessary for detecting larger DNA molecules by mass spectrometry. Oligonucleotides complementary to the internal region of the amplicon are degraded by the 5'-3' exonuclease activity and the degradation products are analyzed by MALDI mass spectrometry. We refer to this assay as the Exo-taq assay or probe degradation assay. This method should be amenable to automation.     

Use of ESI-MS to determine reaction pathway for hydrogen sulphide scavenging with 1,3,5-tri-(2-hydroxyethyl)-hexahydro-s-triazine

To study the reaction between hydrogen sulphide and 1,3,5-tri-(2-hydroxyethyl)-hexahydro-s-triazine, which is an often used hydrogen sulphide scavenger, electro spray ionisation mass spectrometry (ESI-MS) was used. The investigation was carried out in positive mode, and tandem mass spectrometry was used to investigate the nature of unknown peaks in the mass spectra. The reaction was found to proceed as expected from theory with the triazine reacting with hydrogen sulphide to form the corresponding thiadiazine. This species subsequently reacted with a second hydrogen sulphide molecule to form the dithiazine species, hereby confirming previously obtained results and showing the ability of the ESI-MS method for studying the scavenging reaction. The final theoretical product s-trithiane was not detected. Furthermore, fragmentation products of thiadiazine and dithiazine were detected in the solution, and possible pathways and structures were suggested to describe the observed fragments. In these, thiadiazine fragmented to 2-(methylidene amino)-ethanol and 2-(1,3-thiazetidin-3-yl)-ethanol and N-(2-hydroxyethyl)-N-(sulfanylmethyl)-ethaniminium, which underwent a further fragmentation to N-methyl-N-(2-oxoethyl)-methaniminium. Dithiazine fragmented to N-methyl-N-(2-oxoethyl)-methaniminium as well. The by-product from this reaction is methanedithiol, which was not detected due to its low polarity.     

An alternative electrolyte based on acetylacetone–pyridine–iodine for dye-sensitized solar cells

We prepared a new organic electrolyte by the reaction among acetylacetone, pyridine and iodine in 3-methoxypropionitrile. The UV–Visible spectra, conductivity measurement and ESI mass spectra were used to study this electrolyte. It was suggested that the quaternization reaction of pyridine took place in this electrolyte solution and two kinds of pyridinium iodide were formed. The efficiency of dye-sensitized solar cells (DSCs) using this electrolyte reaches 6.72%, which is higher than that of DSCs using LiI electrolyte and methylpropylimidazolium iodide electrolyte. It implies that these pyridinium iodides are effective alternative component of iodide for the electrolytes of DSCs. As this organic iodide electrolyte was in situ synthesized based on iodine instead of alkyl iodide, it will be cost-effective and facilitative for the production of DSCs.     

低浓度甲醛对多肽和蛋白化学修饰的质谱研究

采用基质辅助激光解析电离飞行时间质谱( MALDI-TOF MS)和纳升电喷雾四极杆飞行时间串联质谱( Nano-ESI -QTOF MS)技术,以标准肽段和流感病毒基质蛋白酶切肽段为模型,研究了甲醛对蛋白质和多肽主链的修饰作用。采用与实际病毒灭活过程一致的实验条件(4℃,0.025%(V/V)福尔马林(37%(w/w)甲醛溶液)处理72 h),进行甲醛与多肽的化学反应。结果表明,在实验条件下,甲醛能与标准肽段N端的氨基反应生成羟甲基加合物,再发生缩合反应生成亚胺,形成+12 Da的产物。此外,甲醛还能与标准肽段中的精氨酸、赖氨酸的侧链发生反应,生成+12 Da的反应产物。对流感病毒基质蛋白的酶切肽段与甲醛的反应的质谱分析结果显示,多数的肽段都生成了+24 Da的产物,质量的增加来源于肽段N端氨基(+12 Da)和C端精氨酸或赖氨酸的侧链(+12 Da)的贡献。此外,还观察到有一个漏切位点的肽段生成了+36 Da的产物。本研究结果表明,在实验条件下,低浓度甲醛主要与肽段和蛋白的N 端氨基,以及精氨酸和赖氨酸侧链发生反应。本研究为分析低浓度甲醛与蛋白质的反应产物提供了有效的质谱分析方法和解谱依据。     

Liquid chromatography with complementary electrospray and inductively coupled plasma mass spectrometric detection of ferrocene-labelled peptides and proteins

Succinimidylferrocenyl propionate (SFP) is introduced as labelling agent for amino functions in peptides and proteins. The resulting derivatives are characterised by considerably lower polarity compared with the native analytes and can thus be well separated by means of reversed phase liquid chromatography (RP-LC). The reaction products are characterised by electrospray ionisation mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS). A further advantage of the method is a simple and straightforward derivatisation protocol. Different basic and acidic model proteins as lysozyme, ß-lactoglobulin A and insulin were derivatised using SFP. Furthermore, the first dual-labelling strategy of thiol and amino groups with ferrocene-based reagents is presented. Whereas the amino groups were derivatised with SFP, the thiol groups were functionalised by reaction with ferrocenecarboxylic acid(2-maleimidoyl)ethylamide. Again, LC/ESI-MS is a suitable tool to characterise the modified peptides and proteins.