Effects of Temperature and Energy on Stability of Oligomeric Enzyme Probed on Electrospray Ionization Mass Spectrometry

Escherichia coli 3-Deoxy-D-manno-octulosonate 8-phosphate（KDO8P） synthase catalyzed the condensation reaction between D-arabinose 5-phosphate（A5P） and phosphoenolpyruvate（PEP） to form KDO8P and inorganic phosphate（Pi）. The noncovalent tetrameric association ofKDO8P synthase was observed and dissociated in gas phase by means of electrospray ionization mass spectrometry under the very ＂soft＂ conditions. The results indicate that PEP-bound enzyme generated abundant tetrameric species as well as monomeric species at the ＂soft＂ conditions, whereas, the unbound enzyme favored the formation of a dimeric species. The mass spectra of the mixture of the enzyme with one of substrates, PEP, and A5P or one of products, KDO8P and Pi show that the complex of the unbound enzyme with PEP or Pi was prone to the formation of a monomeric species, whereas, that of the unbound enzyme with A5P or KDO8P was similar to the unbound enzyme. The intensity of the dimeric species increased with the increase of temperature at a collision voltage of 10 V. Taken together, the results presented here suggest that mass spectrometry will be a powerful tool to explore subtile conformational changes and/or subunit-subunit interactions of multiprotein assembly induced by ligand-binding and/or the changes of environmental conditions.     

脱水淫羊藿素的电喷雾多级串联质谱研究

研究了脱水淫羊藿素的电喷雾质谱行为，结果表明：该化合物在正、负离子模式下，均可得到较好的电喷雾质谱信息，且在负离子模式下电喷雾质谱分析的灵敏度较高；运用电喷雾碰撞诱导解离技术，发现正离子和负离子模式下的裂解方式虽有相似之处但并不完全相同；分别阐明了该化合物在正、负离子模式下的电喷雾质谱碎裂规律。     

利用电喷雾质谱研究单链寡聚核苷酸与人参皂苷的相互作用

选择与SARS病毒相关的寡聚核苷酸片段作为靶分子, 利用电喷雾质谱研究人参皂苷与靶分子形成的非共价复合物. 探讨了实验条件对单链寡聚核苷酸靶分子与皂苷类化合物相互作用的影响; 发现非共价作用强度的大小与人参皂苷苷元、糖链的结构有关, 包括人参皂苷苷元的类型、羟基的数目、糖链的长短. 发现人参皂苷与单链寡聚核苷酸靶分子相互作用的强度具有如下规律: 三醇型苷元＞二醇型苷元, 三醇型皂苷＞二醇型皂苷; 在三醇型皂苷中, 三糖＞二糖＞四糖, 一糖＞苷元, 具有相同糖单位的二醇型皂苷的四糖苷中, 分子量小的皂苷＞分子量大的皂苷, 同分异构体中, 同为木糖末端的Rb2＞Rc, 即吡喃型＞呋喃型. 上述研究结果将有助于阐述人参皂苷的药理活性、构效关系.     

芹菜素的电喷雾萃取电离串联质谱

采用实验室自制的电喷雾萃取电离源(EESI),结合串联质谱(MSn)技术,对芹菜素这一典型的黄酮类活性化合物的质谱行为进行了研究。实验表明,在正、负离子检测模式下,该化合物均能得到较好的EESI-MS信号,且在负离子检测模式下灵敏度更高。通过对比芹菜素的EESI-MS和电喷雾电离质谱(ESI-MS)谱图发现,芹菜素在EESI-MS和ESI-MS中的裂解规律相似,但是EESI是一种比ESI更软的电离模式。根据对芹菜素EESI-MS特征碎片离子的分析,提出了芹菜素在EESI-MS中裂解的基本规律,为EESI-MS技术用于分析、鉴定复杂基质中痕量芹菜素奠定了理论和实验基础。     

电喷雾萃取电离质谱法检测1-羟基芘

采用电喷雾萃取电离质谱(EESI-MS)分析致癌性环境有机污染物多环芳烃(PAHs)生物标志物1-羟基芘(1-OHP),探究1-OHP在EESI源中电离的可行性,考察ESI溶剂和样品溶液组成对方法灵敏度的影响,初步建立I-OHP的EESI MS半定量分析方法.结果表明,溶液中1-OHP能够在EES1源中有效电离,生成准分子离子[M-H]- (m/z 217),并得到其二级质谱特征碎片离子[M- H- CO]- (m/z 189);水、甲醇、乙醇、正丙醇和正丁醇5种ESI溶剂中,使用甲醇时,离子峰m/z 217信噪比最大.样品溶液中甲醇含量越高,离子峰m/z 217强度越强.离子峰m/z 217强度与1-OHP浓度在10～200 μg/L内的线性相关性相对最好;相关系数(R)0.982;相对标准偏差(RSD)为3.4％～14.0％(n=5);定量下限约为10 μg/L(S/N=10);单次检测时间小于0.5 min.     

Laser Desorption Ionization Versus Electrospray Ionization Mass Spectrometry: Applications in the Analysis of Cluster Anions

Mass spectra of transition metal carbonyl cluster anions were recorded using laser desorption ionization time-of-flight (LDI-TOF) and electrospray ionization (ESI) techniques. The LDI spectra generally contain peaks corresponding the intact cluster together with extensive CO loss fragments ions whereas the ESI spectra exhibit peaks corresponding the intact cluster together with few (if any) CO loss fragment ions. The parameters of both techniques can be modified to vary the extent of fragmentation. In all cases no fragmentation of the metal core is observed. Overall, ESI is a more informative method for the analysis of these types of cluster anions.     

Toward Depth-Resolved Analysis of Plant Metabolites by Nanospray Desorption Electrospray Ionization Mass Spectrometry

Nanospray desorption electrospray ionization (nano-DESI) is one of the ambient desorption ionization methods for mass spectrometry (MS), and it utilizes a steady-state liquid junction formed between two microcapillaries to directly extract analytes from sample surfaces with minimal sample damage. In this study, we employed nano-DESI MS to perform a metabolite fingerprinting analysis directly from a Hypericum leaf surface. Moreover, we investigated whether changes in metabolite fingerprints with time can be related to metabolite distribution according to depth. From a raw Hypericum leaf, the mass spectral fingerprints of key metabolites, including flavonoids and prenylated phloroglucinols, were successfully obtained using ethanol as a nano-DESI solvent, and the changes in their intensities were observed with time via full mass scan experiments. In addition, the differential extraction patterns of the obtained mass spectral fingerprints were clearly visualized over time through selected ion monitoring and pseudo-selected reaction monitoring experiments. To examine the correlation between the time-dependent changes in the metabolite fingerprints and depth-wise metabolite distribution, we performed a nano-DESI MS analysis against leaves whose surface layers were removed multiple times by forming polymeric gum Arabic films on their surfaces, followed by detaching. The preliminary results showed that the changes in the metabolite fingerprints according to the number of peelings showed a similar pattern with those obtained from the raw leaves over time.     

L-抗坏血酸电喷雾串联质谱行为研究

采用电喷雾串联质谱(ESI-MSn)技术, 结合氢/氘交换方法, 研究了L-抗坏血酸在正、负离子模式下的质谱行为. 实验表明, 电喷雾质谱能够方便地观测到实验条件下L-抗坏血酸的特征裂解方式和相应碎片; 实验发现毛细管温度对L-抗坏血酸的裂解方式产生显著的影响, 从不同毛细管温度下的相应质谱信号强度变化能观察到L-抗坏血酸及其降解产物的热稳定性差异. 因此, 根据串联质谱所观测到的L-抗坏血酸特征碎片离子, 提出了L-抗坏血酸的结构变化及其降解反应的可能机理和基本规律, 为现代质谱技术快速测定复杂基体样品中的痕量抗坏血酸奠定了实验基础.     

Novel Rearrangement of Small Peptides in Electrospray Ionization Tandem Mass Spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is a powerful method for sequencing peptides. A novel fragmentation pattern with the loss of a neutral fragment of 45 Da was observed with the dipeptides, tripeptides,tetrapeptides and pentapeptides containing phenylalanine or histidine residues. A novel rearrangement reaction with the extrusion of a formamide piece was studied and the rearrangement mechanism was proposed and confirmed by deuterium labeling experiments with ESI-MS^n and high-resolution mass spectrometry. These findings are potentially helpful in identifying the specific sequence pattern in the peptide sequencing.     

电喷雾质谱法研究人参皂苷与溶菌酶的相互作用

用电喷雾质谱法研究了溶菌酶与人参皂苷Rg1和Re的非共价相互作用。非共价复合物的解离常数可以由溶菌酶和复合物的峰高直接计算得到。由各个体系得到的溶菌酶与Rg1和Re的一级解离常数KD, 1是一致的，但是二级解离常数KD, 2的差别较大。由此可以推断出复合物的峰高越强，结果的精密度越好。当固定溶菌酶的浓度不变时，复合物的解离常数会随着人参皂苷的浓度的增加而略微的增大。而当固定人参皂苷的浓度时，复合物的解离常数会随着溶菌酶的浓度的增加而减小。计算结果还证明了人参皂苷Rg1与溶菌酶的结合能力大于Re。     

黄连中小檗碱及其同分异构体的电喷雾质谱研究

利用电喷雾多级串联质谱对黄连经水提、醇沉、薄层色谱分离后，得到的组分进行了详细研究，并对其中小檗碱及其同分异构体表小檗碱的分子离子[M ]进行了源内碰撞诱导解离(CID)，在不同源内CID能量(25％，50％)下碰撞诱解离时存在区别，并提出其碎裂机理方面的解释。     

Fast Screening of Chicken Egg Lysozyme in White Wine Products by Extractive Electrospray Ionization Mass Spectrometry

Fast detection of trace lysozyme,one of the most important food allergens in white wine samples,was achieved by extractive electrospray ionization mass spectrometry without sample pretreatment in this ...     

电喷雾萃取电离质谱法用于掺假白酒的快速分析

采用自行研制的电喷雾萃取电离源(EESI)与LTQ-XL质谱仪耦合,并结合主成分分析(PCA)和聚类分析(CA),建立了一种能在无需样品预处理的条件下直接、快速、准确鉴别茅台等八种掺假白酒(混掺水和工业酒精,且通过酒精计调控掺假酒的酒精度与真酒保持一致)的新方法. 同时,利用串联质谱能够对目标组分进行准确鉴定. 研究结果表明,EESI-LTQ-MS检测单个样品的时间小于1 min,且重现性好,PCA区分正确率高达96.5%. 通过设置未知样分析进一步验证了该方法的可行性. 此外,还结合单光子电离飞行时间质谱法(SPI-TOF-MS)对检测谱图进行对比分析. 阐述了EESI和SPI两种电离技术在挥发性有机物分析上具有各自的优势,且两种检测手段具有互补的特点. 为市场上酒类饮品真假的快速鉴别及品质鉴定建立了一个综合的分析方法,对于快速筛选伪劣酒类产品具有非常重要的应用价值.     

中性解吸电喷雾萃取电离质谱法直接检测蜂蜜中的敌敌畏

采用中性解吸电喷雾萃取电离质谱( ND-EESI-MS)技术,在无需样品预处理的条件下,建立了对蜂蜜中敌敌畏直接快速检测的方法。在正离子模式下,敌敌畏质子化离子峰位于 m/z 223,二级特征离子为m/z 109和127。在优化的条件下,以m/z 127的信号强度为定量指标,建立了蜂蜜中敌敌畏残留的定量检测方法。结果表明,在蜂蜜基质中,敌敌畏在5~1000 ng/mL浓度范围内与m/z 127的信号强度线性关系良好,相关系数为0.998,检出限为1.0 ng/mL(S/N=3);蜂蜜中3个加标水平(10,30和400 ng/mL)的敌敌畏的回收率为93.0％~103.0％,精密度(RSDs)小于4.4％。同时采用气相色谱(火焰光度检测器)方法作为对照方法,检测敌敌畏加标蜂蜜样品,结果表明,加标蜂蜜在5~1000 ng/mL浓度范围内与峰面积线性关系良好,相关系数为0.999,检出限为1.6 ng/mL;10,30和400 ng/mL 3个水平加标蜂蜜的回收率为94.9％~110.3％,精密度小于7.6％。     

二肽衍生物的电喷雾质谱研究

基于HIV整合酶核心结构域,合成了以HIV整合酶为靶标的二肽衍生物,采用多级质谱技术（二级、三级）研究二肽衍生物在质谱条件下的化学键断裂途径,发现主要的断裂方式为：氨基与羰基间的NH-CO键的断裂以及N-(苯并噻唑-2-基)甲酰氨基与亚甲基间的CO-C间的断裂。     

Direct Determination of Chlorpyrifos in Honey by Neutral Desorption-Extractive Electrospray Ionization Mass Spectrometry

Neutral desorption-extractive electrospray ionization mass spectrometry was used for the direct, rapid, and quantitative determination of chlorpyrifos in honey without pretreatment. Under the optimized conditions, the limit of detection was 1.64?ng/mL with a linear dynamic range from 10 to 1,000?ng/mL and relative standard deviations from 1.97 to 6.14%. The recoveries of fortified honey samples at 50 and 500?ng/mL were 82.02 and 109.28% with relative standard deviations of 4.02 and 4.34%, respectively. This protocol offers high sensitivity, good precision, rapid analysis, and high specificity in a complex matrix.     

三氟乙酸增强的解吸电喷雾电离质谱成像用于脂质空间分布研究

将三氟乙酸添加到解吸电喷雾电离质谱的喷雾溶剂中,提高了对脂质的成像检测能力,使小鼠脑组织切片中能检测到的脂质种类从59种提高到71种.此外,该方法还可以促进脂质的质子化峰的形成并抑制钠和钾加合物的形成,有利于简化谱图解析过程并实现更好的定量分析.利用该方法实现了小鼠脑组织切片上多种脂质的空间相关性研究,为探索脂质间潜在的代谢途径提供了新手段,在神经学、临床等领域具有巨大的应用前景.     

Elucidation of O-Phosphoryl and N-Phosphoryl Amino Acids by Electrospray Ionization Tandem Mass Spectrometry

Mass spectroscopic characteristics of phosphoryl amino acids were studied in detail by positive and negative electrospray ionization mass spectrometry (ESI-MS) in conjunction with tandem mass spectrometry (MS/MS). Besides N-diisopropyloxyphosphoryl amino acids (N-DIPP-AA), O-phospho- and O-diisopropyloxyphosphoryl amino acids (O-DIPP-AA) were studied and compared to N-DIPP-AA. The fragmentation pathways of [M H]^ ,[M Na]^ and [M-H]^- ions of phosphoryl amino acids were summarized. In addition to several similar patterns, each of them showed its characteristic fragmention.     

雪上一支蒿中乌头碱类生物碱的电喷雾串联质谱分析

利用电喷雾串联质谱(ESI-MS／MS)对雪上一支篙的乙醇提取液进行了直接分析，方法简便，直观，用样量少。ESL-MS可以给出分子量信息，MS／MS方法则可以从复杂体系中获得结构信息。在雪上一支蒿中发现乌头碱、去氧乌头碱及它们的水解产物和脂类生物碱等共19种二菇生物碱，其中脂类生物碱为首次在该植物中发现。