Studies on the Basic Conformation of Lysozyme by Electrospray Ionization-Mass Spectrometry(ESI-MS)

In the present work, 2-mercaptoethanol and dithiothreitol were used to hydrogenise the internal disulfide bond in lysozyme. The experimental results indicate that the charge distribution of the proteins are different in the reaction process. From the calculated molecular weight, the reduction process of the disulfide bond in the molecules can be described, and the number of the disulfide bonds in the molecule can also be determined.

High Performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometric Analysis of Bilobalide and Ginkgolides in Ginkgo biloba L. Leaves

The ginkgo terpenoids including bilobalide and ginkgolides are the main pharmaceutical components in the leaves or extracts of Ginkgo biloba L. In this paper, the analysis of bilobalide and ginkgolides in leaves of Ginkgo biloba L. by high performance liquid chromatography (HPLC)-electrospray ionization (ESI)-mass spectrometry (MS) was carried out. The separation was performed on Inertsil ODS3 column with methanol-water (36:64) as mobile phase, with 1 mL·min -1 of flow rate at 35℃. Then the mass spectrum analysis was conducted by ZMD micromass electrospray ionization (ESI)-mass spectrometer (MS). The HPLC total ion chromatogram and selected ion chromatogram (with 325, 407, 423, 439 of m/z) of the sample and ESI-/MS mass spectra of the peaks in the chromatograms were obtained. So bilobalide, ginkgolide A, B, C and J in Ginkgo biloba L. leaves were identified. The method is easy and rapid, with a good accuracy.

Rapid authentication of different herbal medicines by heating online extraction electrospray ionization mass spectrometry

The rapid and accurate authentication of traditional Chinese medicines(TCMs)has always been a key scientific and technical problem in the field of pharmaceutical analysis.Herein,a novel heating online extraction electrospray ionization mass spectrometry(H-oEESI-MS)was developed for the rapid and direct analysis of extremely complex substances without the requirement for any sample pretreatment or pre-separation steps.The overall molecular profile and fragment structure features of various herbal medicines could be completely captured within 10–15 s,with minimal sample(<0.5 mg)and solvent consumption(<20μL for one sample).Furthermore,a rapid differentiation and authentication strategy for TCMs based on H-oEESI-MS was proposed,including metabolic profile characterization,characteristic marker screening and identification,and multivariate statistical analysis model validation.In an analysis of 52 batches of seven types of Aconitum medicinal materials,20 and 21 key compounds were screened out as the characteristic markers of raw and processed Aconitum herbal medicines,respectively,and the possible structures of all the characteristic markers were comprehensively identified based on Compound Discoverer databases.Finally,multivariate statistical analysis showed that all the different types of herbal medicines were well differentiated and identified(R^(2)X>0.87,R^(2)Y>0.91,and Q^(2)>0.72),which further verified the feasibility and reliability of this comprehensive strategy for the rapid authentication of different TCMs based on H-oEESI-MS.In summary,this rapid authentication strategy realized the ultra-high-throughput,low-cost,and standardized detection of various complex TCMs for the first time,thereby demonstrating wide applicability and value for the development of quality standards for TCMs.

Unraveling enhanced membrane lipid biosynthesis in Chlamydomonas reinhardtii starchless mutant sta6 by using an electrospray ionization mass spectrometry-based lipidomics method

The unicellular green alga Chlamydomonas reinhardtii,a well-established model organism,has been widely used in dissecting glycerolipid metabolism in oxygenating photosynthetic organisms.In previous studies,it has been found that shunting carbon precursors from the starch synthesis pathway can lead to a 10-fold increase in TAG content as compared to the wild type,but it is unknown whether inactivation of AGPase may affect membrane lipids biosynthesis.The study aims to investigate global changes in lipid metabolism and homeostasis in the starchless mutant C.reinhardtii sta6.By utilizing an electrospray ionization/mass spectrometry(ESI/MS)-based lipidomics approach,a total of 105 membrane lipid molecules of C.reinhardtii were resolved,including 16 monogalactosyldiacylglycerol(MGDG),16 digalactosyldiacylglycerol(DGDG),11 phosphatidylglycerol(PG),6 sulfoquinovosyldiacylglycerol(SQDG),49 diacylglyceryl-N,N,N-trimethylhomoserine(DGTS),2 phosphatidylethanolamine(PE),and 5 phosphatidylinositol(PI)molecules.The quantitative results indicated that the membrane lipid profiles were similar between the two C.reinhardtii strains grown under both low-and high-light conditions,but the cellular contents of a great number of lipids were altered in sta6 due to the defect in starch biosynthesis.Under low-light conditions,sta6 accumulated more PI,MGDG,DGDG but less amounts of DGTS as compared to WT.Under high light,sta6 cells contained higher content membrane lipids than cc-124,except for PG,which is more or less similar in both strains.Our results demonstrate that the cellular membrane lipid homeostasis underwent profound changes in the starchless mutant,and thereby its physiological impact remains to be explored.

Liquid Chromatography-electrospray Ionization Tandem Mass Spectrometry for Simultaneous Determination of Metformin and Glimepiride in Beagle Dog Plasma and Bioequivalence Study

A sensitive and selective liquid chromatography-electrospray ionization tandem mass spectrometry（LC- ES1-MS/MS） was used for the simultaneous determination of metformin and glimepiride in beagle dog plasma with glipizide as internal standard（IS）. After simplified protein precipitation with methanol, both the analytes and IS were chromatographed on a Zorbax CN column via gradient elution with methanol（containing 5 mmol/L ammonium ace- tate） and 5 mmol/L aqueous ammonium acetate as the mobile phase. Detection was performed by multiple reaction monitoring（MRM） scanning via ESI source operated in positive ionization mode. Specificity, linearity, accuracy, pre- cision, recovery, matrix effect and stability were validated for metformin and glimepiride in beagle dog plasma. The calibration curves were linear in a concentration range of 10-10000 ng/mL for metformin and 4-4000 ng/mL for glimepiride with both correlation coefficients higher than 0.99. The recoveries obtained for the analytes and IS were all between 82.7% and 101.2%. The method exhibited excellent performance in terms of selectivity, robustness, short analytical time and simplicity of sample preparation. Finally, the proposed method was applied to a bioequivalence study of self-made bilayer tablet and commercial formulation containing 500 mg of metformin and 1 mg of glimepi- ride in beagle dogs.

Studies on Fragmentation Pathways of N-Ethoxy(phenyl) phosphoryl Amino Acids by Electrospray Ionization Tandem Mass Spectrometry

The positive and negative ESI-MS/MS spectra of N-ethoxy（phenyl） phosphoryl amino acids（EPP-AA） were investigated by electrospray ionization（ESI） ion trap mass spectrometry. The fragmentation pathways of [ M ＋ Na]^＋ and [ M - H]^- ions are proposed and rationalized. The observation may have some potential applications in the interpretation of the MS/MS spectra of novel N-phosphoryl compounds. The complexity of MS/MS spectra of EPP-AA [ M ＋ Na]^＋ ions is decreased compared with that of N-dialkyloxyphosphoryl amino acid. Therefore, the new phosphonamidate method may be considered one of the superior methods that can be used in sequencing peptides and proteins extensively.

Studies on Fragmentation of Peptide by Nano-electrospray Ionization Fourier Transform Ion Cyclotron Resonance Multi-stage Tandem Mass Spectrometry

The sequence analysis of peptides was performed by nano-electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry（Nano-ESI-FT-ICR-MSn） and several peptides were chosen as examples. With the aid of the collision induced dissociation（CID）, FT-ICR provides not only precise mass/charge ratio, but also structure information of the selected peptides. The fragment ions were identified according to the observed molecular weights and peptide sequence was determined successfully. So Nano-ESI-FT-ICR-MSn is a useful tool for identification of the amino acid sequence of peptides with high confidence. Besides, a pathway for the dehydration of y ions without amino acids containing carboxylic acid under sustained off-resonance irradiation collision-induced dissociation（SORI-CID） condition was proposed.

Identification,structure elucidation and origin of a common pyridinium-thiocyanate intermediate in electrospray mass spectrometry among the benziamidazole-class proton pump inhibitors

During the analysis of benziamidazole-class irreversible proton pump inhibitors,an unusual mass spectral response with the mass-to-charge ratio at[Mt10]t intrigued us,as it couldn't be assigned to any literature known relevant structure,intermediate or adduct ion.Moreover,this mysterious mass pattern of[Mt10]t has been gradually observed by series of marketed proton pump inhibitors,viz.omeprazole,pantoprazole,lansoprazole and rabeprazole.All the previous attempts to isolate the corresponding component were unsuccessful.The investigation of present work addresses this kind of signal to a pyridinium thiocyanate mass spectral intermediate(10),which is the common fragment ion of series of labile aggregates.The origin of such aggregates can be traced to the reactive intermediates formed by acid-promoted degradation.These reactive intermediates tend to react with each other and give raise series of complicated aggregates systematically in a water/acetonitrile solution by electrospray ionization.The structure of the corresponding pyridinium thiocyanate species of omeprazole(10a)has been eventually characterized with the help of synthetic specimen(10a′).Our structural proposal as well as its origin was supported by in situ nuclear magnetic resonance,chemical derivatization and colorimetric experiments.

Analysis of Norditerpenoid Alkaloids Extracted from Aconitum sinomantanum Nakai by Electrospray Ionization Tandem Mass Spectrometry

Electrospray ionization mass spectrometry（ESI-MS） was applied simultaneously in determining norditerpenoid alkaloids from the roots of Aconitum sinomantanum Nakai （RAS） based on molecular mass information. The tandem mass spectra（ ESI-MS^n） provided the alkaloidal structural information, through which the existence of these alkaloids was further confirmed. Accordingly, six known norditerpenoid alkaloids were simultaneously determined on the basis of their ESI-MS^n spectra. Furthermore, based on the diagnostic fragmentation pathways of alkaloidal MS^n, a rapid method for direct detection and characterization of alkaloids from an ethanolic extract of RAS was described.

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 study.The multiply-charged ions of m/z1587 were chosen for the quantitative detection of lysozyme in white wine,showing linear dynamic signal responses in a range of 5―75μg/mL with a linearity coefficient of 0.999 and an acceptable relative standard deviation（RSD） of 8.0%―15.0% for directly measuring lysozyme in the complex food samples.The limit of detection for lysozyme in white wine sample was calculated to be 5 μg/mL,which was lower than the amounts that can provoke allergic reactions（oral test with 3 mg or labial test with 1 mg/mL）.A single sample analysis was completed within 1 min.The data demonstrate that extractive electrospray ionization mass spectrometry is a useful tool for fast screening lysozyme in the complex matrix,showing promising application in the rapid detection of food allergen.

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.

Molecular Probes in Tandem Electrospray Ionization Mass Spectrometry: Application to Tracing Chemical Changes of Specific Phospholipid Molecular Species

New ionization and detection techniques in mass spectrometry have been successfully applied for efficient analyses of complex biological systems. It is, however, still difficult to trace structural changes of a specific molecular species in such systems. In the present study, a molecular probe strategy in combination with tandem electrospray ionization mass spectrometry has been examined using synthetic deuterium-labeled phosphatidylcholine hydroperoxide (PC-OOH/D3) and ethyl-labeled phosphatidylcholine having docosahexaenoic acid side chain (DHA-PC/Et). Administration of a mixture of PC-OOH/D3 and DHA-PC/Et to human blood and human skin surface, followed by extraction and analysis with collision-induced tandem electrospray ionization mass spectrometry demonstrated that metabolites of both molecular probes can be detected simultaneously with strict selectivity. The present method is also found to be useful in tracing chemical changes of the unstable docosahexaenoyl group on the surface of processed fish. The activity of phospholipase A2 can also be assessed using a phospholipid molecular probe with a linoleoyl and a deuteriomethyl group via selective detection of the lyso-phospholipid product by mass spectrometry. The advantage of the present method is that no chromatographic separation is required and analysis can be performed under strictly the same condition for different molecular probes, affording multiple data by one experiment. The present strategy may be useful for tracing time-dependent phenomena in dynamic phospholipid biochemistry, and can be widely used for any biological and food systems.

ESI-MS研究六钼酸盐芳香亚胺衍生物的合成反应及气相稳定性

该文通过合成两种Lindqvist型六钼酸芳香亚胺含氯衍生物TBA_(2)[Mo_(6)O_(18)(N-C_(6)H_(4)-Cl-p)](Ⅰ)和TBA_(2)[Mo_(6)O_(18)(N-C_(6)H_(4)-Cl-o)](Ⅱ),并采用电喷雾质谱法(ESI-MS)实时监测其合成反应过程,对两个反应体系中各物质含量随反应时间的变化趋势进行分析,考察了多酸芳香亚胺衍生物中芳香胺不同取代基的位置对衍生物合成反应速率及结构稳定性的影响。并通过碰撞诱导解离技术(CID)研究了两种衍生物离子的气相裂解反应,获得了其气相裂解途径及结构稳定性信息。ESI-MS实时监测结果显示,两种衍生物合成过程中,0~3 h内产物生成速率较小,而3~6 h期间反应速率最大,6~9 h次之,9~12 h反应速率明显减小。对氯苯胺的pKb小于邻氯苯胺,是衍生物Ⅰ的合成反应速率大于Ⅱ的重要原因。CID结果表明,相较于Ⅰ离子,Ⅱ离子的气相结构稳定性更弱,主要原因是Cl原子取代基的综合电子效应为吸电子效应,其在邻位取代比对位取代距离维系离子稳定性的Mo≡N键更近,导致Ⅱ中Mo≡N键电子云密度下降程度更大,离子的稳定性比Ⅰ更弱。综上,ESI-MS是一种研究多酸有机官能化衍生物的可靠技术,可为该类衍生物结构稳定性的研究及后期的导向设计合成提供基础信息。

Comparison of different approaches for direct coupling of solid-phase microextraction to mass spectrometry for drugs of abuse analysis in plasma

The direct coupling of solid-phase microextraction(SPME)to mass spectrometry(MS)(SPME-MS)has proven to be an effective method for the fast screening and quantitative analysis of compounds in complex matrices such as blood and plasma.In recent years,our lab has developed three novel SPME-MS techniques:SPME-microfluidic open interface-MS(SPME-MOI-MS),coated blade spray-MS(CBS-MS),and SPME-probe electrospray ionization-MS(SPME-PESI-MS).The fast and high-throughput nature of these SPME-MS technologies makes them attractive options for point-of-care analysis and anti-doping testing.However,all these three techniques utilize different SPME geometries and were tested with different MS instruments.Lack of comparative data makes it difficult to determine which of these methodologies is the best option for any given application.This work fills this gap by making a comprehensive comparison of these three technologies with different SPME devices including SPME fibers,CBS blades,and SPME-PESI probes and SPME-liquid chromatography-MS(SPME-LC-MS)for the analysis of drugs of abuse using the same MS instrument.Furthermore,for the first time,we developed different desorption chambers for MOI-MS for coupling with SPME fibers,CBS blades,and SPME-PESI probes,thus illustrating the universality of this approach.In total,eight analytical methods were developed,with the experimental data showing that all the SPME-based methods provided good analytical performance with R^(2)of linearities larger than 0.9925,accuracies between 81%and 118%,and good precision with an RSD%≤13%.

一例三(5-氯苯并噻唑-2-甲基)胺Fe(Ⅱ)配合物的串联合成过程及电催化性质研究

三(芳基甲基)胺由于强配位性,在氧气活化的单/双金属非血红素金属蛋白类似物的合成中起着重要作用,且能够作为加速配体催化Click反应,因而受到广泛关注。本研究以苯并[d]噻唑-2-基甲胺(S1)和5-氯苯并[d]噻唑-2-基)甲醇(L1)为原料,在FeCl_(3)·6H_(2)O的催化下,串联合成得到了一例新颖的三(5-氯苯并噻唑甲基)胺Fe(Ⅱ)配合物1。用X-射线单晶衍射仪、X-射线粉末衍射仪、电喷雾离子化质谱确证了结构,通过晶体学和电喷雾离子质谱相结合,研究了反应过程,检测到了一系列关键中间体片段([i-Cl]^(+)、[ii+H]^(+)、[iii-Cl]^(+)),结合反应过程跟踪提出了可能的反应机理,包括N-烷基化,氧化水解等步骤,实现了3个共价键的连续构筑。对配合物1进行了OER(析氧反应)测试及稳定性性能研究,其中过电势为391 mV,Tafel斜率为95.9 mV·dec^(-1),在电极密度10 mA/cm^(2)下进行了10 h的恒电流电解,在该时间范围内稳定性良好。通过对配合物1的电催化性能研究则为后续该配体的应用领域提供一种新的方向。

非变性电喷雾质谱研究二甲基亚砜对溶菌酶-黄酮复合物的影响

电喷雾质谱(ESI-MS)在研究蛋白质与小分子相互作用方面有着重要应用,然而,添加剂的加入会直接影响ESI-MS分析。目前,二甲基亚砜(DMSO)对质谱分析蛋白质-小分子相互作用的影响尚不明确。本文探究了DMSO对淫羊藿苷、芦丁、柚皮苷、野黄芩苷与溶菌酶复合物的质谱分析影响。发现DMSO不仅影响溶菌酶复合物的表观结合常数和电荷态,还在一定程度上增加了溶菌酶-黄酮复合物的表观亲和力。与不添加DMSO相比,DMSO加入量较低时,复合物的电荷态降低;DMSO加入量略高时,复合物的电荷态升高,且高于未加DMSO时的电荷态。研究表明,DMSO可以用于稳定ESI-MS中的溶菌酶-黄酮复合物,但应控制DMSO用量。

无鞘流毛细管电泳-电喷雾串联质谱用于面粉中荧光增白剂的高灵敏检测

食品中残留的过量荧光增白剂(FWAs)对人体健康存在潜在威胁,因此,开发准确、高灵敏的FWAs检测方法在食品安全监测方面具有重要意义。本工作提出了利用无鞘流接口的毛细管电泳-电喷雾串联质谱(CE-ESI-MS/MS)的方法,以实现面粉样品中6种FWAs的高灵敏检测。实验采用超声辅助液相萃取法进行样品前处理,以减小复杂样品中基质效应的干扰。以氯仿-甲醇(3∶2,v/v)溶液作为萃取剂,在30℃下对样品中的FWAs进行萃取。萃取完成后,经离心、氮气吹干后用氯仿-甲醇(1∶4,v/v)复溶后检测。无鞘流CE-ESI-MS/MS方法采用正离子(ESI+)和多反应监测(MRM)模式,利用二级质谱对6种目标物同时进行定性定量分析,从而提高方法的检测通量和灵敏度。结果显示,本方法具有较宽的线性范围、良好的线性关系和较低的方法检出限(0.04~0.67 ng/g),在实际样品中3个水平下的加标回收率良好(86.2%~103.7%),日间和日内重复性(RSD)分别不大于11.5%和10.2%。上述研究表明,本方法适用于复杂基质中多种FWAs的准确、高灵敏检测,在面粉样品的质量评估和FWAs的污染监控方面具有潜在的应用价值。

Simultaneous determination of seven flavonoids in Epimedium by liquid chromatography-tandem mass spectrometry method

In this paper, a sensitive and specific liquid chromatography-electrospray ionisation-mass spectrometry （LC-ESI-MS） method has been developed and validated for the identification and determination of seven flavonoids, namely epimedin A, epimedin B, epimedin C, icariin, sagittatoside B, 2＂-O-rhamnosyl icariside II, and baohuoside I in Epimedium from different sources.

电喷雾萃取电离-串联质谱法测定不同水样中全氟辛酸

全氟辛酸(PFOA)是一种持久性有机污染物,长期环境暴露会对生态系统与人类健康造成极大的风险,因此,发展快速定量监测水中PFOA的方法至关重要。本研究采用电喷雾萃取电离-串联质谱(EESI-MS/MS)法检测自来水、江水、河水中的痕量PFOA。结果表明,在优化的实验条件下,PFOA在1~500 ng/L浓度范围内的线性关系良好,线性相关系数(R^(2))为0.997 6,方法检出限(LOD)为0.57 ng/L,定量限(LOQ)为1.73 ng/L,回收率为92.1%~105.2%,相对标准偏差(RSD,n=6)为1.89%~5.45%。本方法具有灵敏度高、准确性好、分析速度快等优点,在监测环境水体污染物方面具有良好的应用前景。

电喷雾萃取电离-串联质谱测定鲜竹沥中的二苯并[a,h]蒽

采用电喷雾萃取电离-串联质谱(EESI-MS/MS)技术,在优化条件下建立了鲜竹沥中二苯并[a,h]蒽(DBA)的快速检测方法。在正离子检测模式下,对离子传输管温度、喷雾电压、电喷雾溶剂流速、样品流速进行优化。以硝酸银的甲醇溶液为电喷雾溶剂,诱导DBA和Ag+形成[DBA+Ag]+、[2DBA+Ag]+等特征离子,再结合碰撞诱导解离技术,最终实现了鲜竹沥中DBA的快速定性定量检测。实验结果表明,DBA在0.1~100μg/L质量浓度范围内线性良好,线性相关系数(r2)为0.998 0,方法检出限(LOD)为0.054μg/L,定量下限(LOQ)为0.179μg/L。该方法对鲜竹沥中DBA的加标回收率为81.5%~87.1%,相对标准偏差为1.9%~3.9%。所建方法具有无需复杂样品预处理、准确度高、可直接快速检测等优点,适用于鲜竹沥中DBA的快速筛查与直接分析,同时也为其他PAHs的检测提供了技术手段。